Textbook of Aging Skin
Miranda A. Farage, Kenneth W. Miller and Howard I. Maibach Editors
Textbook of Aging Skin With 366 Figures and 156 Tables
Editors: Miranda A. Farage, Ph.D. Principal Scientist The Procter & Gamble Company 6110 Center Hill Avenue Cincinnati, OH 45224 USA Kenneth W. Miller, Ph.D. Associate Director The Procter & Gamble Company 6110 Center Hill Avenue Cincinnati, OH 45224 USA Howard I. Maibach, M.D. Professor Department of Dermatology University of California, School of Medicine San Francisco, CA 94122 USA
Library of Congress Control Number: 2009938632 ISBN 978-3-540-89655-5 This publication is available also as: Electronic publication under ISBN 978-3-540-89656-2 Print and electronic bundle under ISBN 978-3-540-89935-8 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. ß Springer-Verlag Berlin Heidelberg 2010 The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publishers cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. Springer is part of Springer Science+Business Media www.springer.com Publishing Editor: Tobias Kemme MRW Editor: Sandra Fabiani Printed on acid‐free paper
Dedicated to those on a forgotten and sometimes lonely and scary aging journey—much more dignity and respect are deserved for all of you. – MAF, KWM and HIM
When things go wrong as they sometimes will, When the road you’re trudging seems all uphill, When the funds are low and the debts are high, And you want to smile but you have to sigh, When care is pressing you down a bit Rest if you must, but don’t you quit. Success is failure turned inside out, The silver tint on the clouds of doubt, And you can never tell how close you are, It may be near when it seems afar. So, stick to the fight when you’re hardest hit It’s when things go wrong that you mustn’t quit. —Unknown
Acknowledgments
Deep appreciation and grateful thank-yous are extended to the significant efforts of many people who contributed both knowingly and indirectly to this book, by dedicating their valuable time in preparing their chapters. This book represents the fruits of a jointly conceived and executed venture, and has also benefited from global and diverse partners. A special thank-you to Dr. Mark Dato and Mr. Ron Visscher for generously offering their time and expertise in peerreviewing the relevant chapters and extending their immense support to this book. No praise is excessive for their efforts, and our heartfelt gratitude goes to them. We would like to single out Mr. Anil Joseph Chandy (Springer Reference Editorial Office) for a special recognition of his great effort, time, discipline, and dedication in moving this book forward in a timely and organized manner. We extend our appreciation to Ms. Marion Philipp and Ms. Ellen Blasig (Springer Heidelberg) too for the same. Last but not least, we acknowledge the assistance provided by Dr. Deborah A. Hutchins, Ms. Zeinab Schwen, Ms. Wendy Wippel, Ms. Gayle Entrup, Ms. Jan Tremaine, Ms. Peggy Firth, and Dr. T. L. Nusair for this book. Their collective recommendations and input have vastly improved the texts assembled here. Above all, we extend our everlasting gratitude and love to our parents, who inspired us and to our families and children, who supported and encouraged us all the way with their incredible patience. Only their continuous care, unconditional love, and incomparable sacrifice made all this possible, and easy to achieve. Miranda A. Farage Kenneth W. Miller Howard I. Maibach
Cincinnati and San Francisco October 2009
Foreword
The population is aging rapidly. Centenarians are no longer a rarity. The fastest growing segment of the population in the United States is people over 80. In the next 25 years, half of the population in the United States will be aged over 50. These shifts will have a tremendous impact on the delivery of healthcare to the elderly and will require a new awareness of how cutaneous disorders affect the quality of life, comprising a heavy burden on health and wellbeing. Physicians and healthcare workers are woefully ignorant of the distress, discomfort, and anxieties of people afflicted by disorders of the skin. There exists a widespread misconception that skin disorders are simply cosmetic nuisances that can be self-treated by a great assortment of anti-aging creams and lotions available at the local drug store. Most of these include high-sounding ingredients such as antioxidants, vitamins, nutrients, botanicals, and ancient folkloristic remedies, the efficacy and safety of which have never been tested. They offer little more than hope in a bottle. The fact is that common skin diseases may not often be lethal but can ruin enjoyment of life. Chronic itchy rashes can be maddening, lowering one’s self-esteem, embarrassing, interfering with sleep, and often accompanied by depression, social isolation, and deterioration of appearance; they can also be uncomfortable, and, not least, costly to treat. The elderly commonly take 15–20 oral supplements daily to fight the ailments of old age. These are generally useless and may be harmful, often interacting adversely with prescription drugs. The elderly often resort to alternative medicines instead of seeing their doctor to obtain FDA-approved drugs, and also often skip their daily doses to save money. Noncompliance is common. Misdiagnosis and mistreatment of the elderly by health-care workers are common. National surveys show that skin diseases increase steadily throughout our lifespan. Old people may have as many as 5–10 coexistent cutaneous problems that are worthy of medical attention. Moreover, the clinical manifestations of skin diseases in the aged often have different appearances than in the young, confounding diagnosis. Importantly, healing of chronic lesions, especially ulcers, is impaired in the elderly. Immunity is weakened, increasing susceptibility to infections. Response to treatment is slower, leading to noncompliance. Adverse drug reactions are common and too commonly not suspected. Management of chronic conditions is difficult and frustrating. The above litany of problems makes this textbook edited by Farage, Miller, and Maibach a welcome addition to the literature. It is invaluable as a reference resource covering exhaustively an enormous number of clinical conditions. No topic is neglected including cosmetic treatments. The numerous contributions are by highly qualified experts who have a published record of expertise. This comprehensive volume is also practical and relevant to the everyday world of clinical practice. The information will be useful to physicians, manufacturers of drugs and skincare products, educators, investigators, nursing home personnel, estheticians, and federal regulators. This first edition is up-to-date, including much new material that belongs to the shelves of every library, which deals with geriatric problems. Dermatologists especially will be remiss if they do not put this volume within easy reach for consultation as they encounter a swelling clientele of aging patients. Albert M. Kligman M.D., Ph.D. Professor Emeritus University of Pennsylvania Philadelphia, PA USA
Preface
The skin is a portal of knowledge on aging. From its softness and smoothness in infancy, through its suppleness in youth, to its wrinkled texture in elders, the skin displays the most visible and accessible manifestations of aging. Due to falling birth rates and rising life expectancies in industrialized countries, the average age of the population is increasing. Research interest in the process of aging has grown and people are becoming obsessed with looking and “staying” young. Although excellent compendia exist on the subject of aging skin, the body of knowledge is burgeoning. Consequently, this handbook compiles information into one comprehensive reference. It covers a range of topics, from the basics of skin structure and function, to the cellular and molecular mechanisms of aging, to the latest bioengineering instruments used to assess age-related changes in the skin. The Nobel Prize in Physiology and Medicine awarded in 2009 to Drs. E. H. Blackburn, C. W. Greider and J. W. Szostak will stimulate research that will ameliorate the effects of aging on the organ systems of both humans and animals. This textbook will simplify approaches when the skin may be an efficient approach to aging based on Dr. Blackburn’s team research. The skin approachability and the opportunities to work on humans will provide us in the near future with rapid therapeutic and preventive applications. Contributors are internationally recognized experts from multiple disciplines germane to this topic. We gratefully acknowledge all contributors for sharing their time and expertise. We expect this handbook to be valuable to researchers and students with an interest in aging skin. Because research progress in this area is so rapid, we hope to update this compendium periodically as advances in the field dictate. The editors welcome suggestions for the second edition. Miranda A. Farage, Kenneth W. Miller, and Howard I. Maibach October 2009
Table of Contents
Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiii List of Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxv
Part 1
Basic Sciences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1
Skin Aging in Animal Models: Histological Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Tapan K. Bhattacharyya
2
Histology of Microvascular Aging of Human Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Peter Helmbold
3
Basophilic (Actinic) Degeneration of the Dermis: An Easy Histological Scoring Approach in Dermal Photoaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Peter Helmbold
Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4
Degenerative Changes in Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Miranda A. Farage . Kenneth W. Miller . Howard I. Maibach
5
Skin Aging: A Brief Summary of Characteristic Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Christina Raschke . Peter Elsner
6
Physiological Variations During Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Ge´rald E. Pie´rard . Philippe Paquet . Emmanuelle Xhauflaire-Uhoda . Pascale Quatresooz
7
The Stratum Corneum and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Anthony V. Rawlings
8
The Importance of Extracellular Matrix Protein 1 as Basement Membrane Protein in Maintaining Skin Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Sandy Sercu . Noritaka Oyama . Joseph Merregaert
9
Pathomechanisms of Endogenously Aged Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Evgenia Makrantonaki . Christos C. Zouboulis
10
Pathomechanisms of Photoaged Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Jean Krutmann
xiv
Table of Contents
11
Proteoglycans in Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Franc¸ois-Xavier Maquart . Ste´phane Bre´zillon . Yanusz Wegrowski
12
Possible Involvement of Basement Membrane Damage by Matrix Metalloproteinases and Serine Proteinases in Skin Aging Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Satoshi Amano
13
Aging and Intrinsic Aging: Pathogenesis and Manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Hanan Assaf . Mohamed A. Adly . Mahmoud R. Hussein
14
Buffering Capacity Considerations in the Elderly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Jacquelyn Levin . Howard I. Maibach
15
Neurotrophins and Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Mohamed A. Adly . Hanan Assaf . Mahmoud R. Hussein
16
Considerations for Thermal Injury: The Elderly as a Sensitive Population . . . . . . . . . . . . . . . . . 159 Donald L. Bjerke
17
Skin Reactivity of the Human Face: Functional Map and Age Related Differences . . . . . . . . . . . 173 Slaheddine Marrakchi . Howard I. Maibach
18
Cluster of Differentiation 1d (CD1d) and Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Mohamed A. Adly . Hanan Assaf . Mahmoud R. Hussein
19
Aging of Epidermal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Alexandra Charruyer . Ruby Ghadially
20
Adipose-derived Stem Cells and their Secretory Factors for Skin Aging . . . . . . . . . . . . . . . . . . . 201 Byung-Soon Park . Won-Serk Kim
21
Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Stacy S. Hawkins . William Shingleton . Jean Adamus . Helen Meldrum
22
Hyaluronan and the Process of Aging in Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 Robert Stern
23
Changes in Nail in the Aged . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 Nelly Rubeiz . Ossama Abbas . Abdul Ghani Kibbi
Specialized Skin: Genital . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 24
Vaginal Secretions with Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Paul R. Summers
25
Unique Skin Immunology of the Lower Female Genital Tract with Age . . . . . . . . . . . . . . . . . . . 253 Paul R. Summers
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26
Aging Genital Skin and Hormone Replacement Therapy Benefits . . . . . . . . . . . . . . . . . . . . . . . . 257 William J. Ledger
Rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 27
Facial Skin Rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Ge´rald E. Pie´rard . Fre´de´rique Henry . Pascale Quatresooz
Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 28
Pathology of Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 Qunshan Jia . J. Frank Nash
Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 29
Alterations of Energy Metabolism in Cutaneous Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 Thomas Blatt . Horst Wenck . Klaus-Peter Wittern
30
Cellular Energy Metabolism and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 Regina Hourigan
Molecular Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 31
DNA Damage and Repair in Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 Daniel B. Yarosh
32
Fibulin-5 Deposition in Human Skin: Decrease with Aging and UVB Exposure and Increase in Solar Elastosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 Satoshi Amano
Endocrinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 33
Sebum Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Claudine Pie´rard-Franchimont . Pascale Quatresooz . Ge´rald E. Pie´rard
34
Climacteric Aging and Oral Hormone Replacement Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . 353 Pascale Quatresooz . Claudine Pie´rard-Franchimont . Ge´rald E. Pie´rard
35
Biological Effects of Estrogen on Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 Zack Thompson . Howard I. Maibach
Stratum Corneum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 36
Corneocyte Size and Cell Renewal: Effects of Aging and Sex Hormones . . . . . . . . . . . . . . . . . . . 371 Enzo Berardesca . Joachim Fluhr
37
Stratum Corneum Cell Layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377 Hachiro Tagami
xv
xvi
Table of Contents
38
Aging and Melanocytes Stimulating Cytokine Expressed by Keratinocyte and Fibroblast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 Mutsumi Okazaki
39
Cyanoacrylate Skin Surface Strippings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Claudine Pie´rard-Franchimont . Jorge Arrese-Estrada . Pascale Quatresooz . Ge´rald E. Pie´rard
40
Biology of Stratum Corneum: Tape Stripping and Protein Quantification . . . . . . . . . . . . . . . . . . 401 Ali Alikhan . Howard I. Maibach
Endogenous and Exogenous Factors in Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . 409 41
Effect of Ozone on Cutaneous Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411 Giuseppe Valacchi
42
Infrared A-induced Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421 Peter Schroeder . Jean Krutmann
43
Global Warming and its Dermatologic Impact on Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . 427 Young Hui . Haw-Yueh Thong . Howard I. Maibach
44
Skin Photodamage Prevention: State of the Art and New Prospects . . . . . . . . . . . . . . . . . . . . . 429 Denize Ainbinder . Elka Touitou
45
Environmental and Genetic Factors in Facial Aging in Twins . . . . . . . . . . . . . . . . . . . . . . . . . . . 441 David J. Rowe . Bahman Guyuron
46
Tobacco Smoke and Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447 Akimichi Morita
Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451 47
DNA Biomarkers in Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453 Kimberly G. Norman . Alex Eshaghian . James E. Sligh
In vitro Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 48
The Use of Reconstructed Skin to Create New In Vitro Models of Skin Aging with Special Emphasis on the Flexibility of Reconstructed Skin . . . . . . . . . . . . . . . . . . . . . . . . . 461 Daniel Asselineau . Sylvie Ricois . Herve´ Pageon . He´le`ne Zucchi . Sole`ne Mine . Sarah Girardeau . Flore Nallet . Se´verine Teluob . Gae¨lle Claviez-Homberg
49
In vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477 Akira Date . Tomohiro Hakozaki
50
Aging of Skin Cells in Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487 Suresh I. S. Rattan
Table of Contents
Pigmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493 51
Hyperpigmentation in Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495 Tomohiro Hakozaki . Cheri L. Swanson . Donald L. Bissett
52
Pigmentation in Ethnic Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503 Rupa Pugashetti . Howard I. Maibach
53
The New Face of Pigmentation and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509 John Nip . S. Brian Potterf . Sheila Rocha . Shilpa Vora . Carol Bosko
Part 2
Disease State/Conditions with Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523
Diseases Associated with Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525 54
Non-neoplastic Disorders of the Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527 Miranda A. Farage . Kenneth W. Miller . Enzo Berardesca . Howard I. Maibach
Malignant Skin Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543 55
Neoplastic Skin Lesions in the Elderly Patient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545 Miranda A. Farage . Kenneth W. Miller . Enzo Berardesca . Howard I. Maibach . Isaac M. Neuhaus
56
Carcinogenesis: UV Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567 Douglas E. Brash . Timothy P. Heffernan . Paul Nghiem
57
Melanoma and Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579 Salina M. Torres . Marianne Berwick
58
Aging-associated Non-melanoma Skin Cancer: A Role for the Dermis . . . . . . . . . . . . . . . . . . . . 587 Davina A. Lewis . Jeffrey B. Travers . Dan F. Spandau
59
Non-surgical Modalities of Treatment for Primary Cutaneous Cancers . . . . . . . . . . . . . . . . . . . . 601 Ossama Abbas . Salah Salman
60
Sunlight Exposure and Skin Thickness Measurements as a Function of Age: Risk Factors for Melanoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609 Panthea Heydari . Andia Heydari . Howard I. Maibach
Non-Malignant Skin Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617 61
Influence of Race, Gender, Age, and Diabetes on the Skin Circulation . . . . . . . . . . . . . . . . . . . . 619 Jerrold Scott Petrofsky . Gurinder Singh Bains
62
Atopic Dermatitis in the Aged . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639 Alexandra Katsarou . Melina C. Armenaka
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63
Dry Skin in Diabetes Mellitus and in Experimental Models of Diabetes . . . . . . . . . . . . . . . . . . . 653 Shingo Sakai . Hachiro Tagami
64
Cutaneous Effects and Sensitive Skin with Incontinence in the Aged . . . . . . . . . . . . . . . . . . . . . 663 Miranda A. Farage . Kenneth W. Miller . Enzo Berardesca . Howard I. Maibach
Part 3
Techniques and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 673
Bioengineering Methods and Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 675 65
Bioengineering Methods and Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 677 Francesca Giusti . Stefania Seidenari
66
Hydration of the Skin Surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687 Hachiro Tagami
67
Transepidermal Water Loss and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 695 Ali Alikhan . Klaus-Peter Wilhelm . Fatima S. Alikhan . Howard I. Maibach
68
Corneocyte Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705 Tetsuji Hirao
69
The Structural and Functional Development of Skin During the First Year of Life: Investigations Using Non-invasive Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 715 Georgios Stamatas
70
Structure of Stratum Corneum Lipid Studied by Electron Paramagnetic Resonance . . . . . . . . . . 725 Kouichi Nakagawa
71
Molecular Concentration Profiling in Skin Using Confocal Raman Spectroscopy . . . . . . . . . . . . 735 Jonathan M. Crowther . Paul J. Matts
72
The Measurement and Perception of Uneven Coloration in Aging Skin . . . . . . . . . . . . . . . . . . . 749 Paul J. Matts
Quality of Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755 73
Assessing Quality of Life in Older Adult Patients with Skin Disorders . . . . . . . . . . . . . . . . . . . . 757 Miranda A. Farage . Kenneth W. Miller . Susan N. Sherman . Joel Tsevat
Percutaneous Penetration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769 74
Percutaneous Penetration of Chemicals and Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771 Michael F. Hughes
75
Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781 Sara Flores . Farzam Gorouhi . Howard I. Maibach
Table of Contents
Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787 76
Skin Aging: A Generalization of the Micro-inflammatory Hypothesis . . . . . . . . . . . . . . . . . . . . . 789 Paolo U. Giacomoni . Glen Rein
Probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797 77
The Potential of Probiotics and Prebiotics for Skin Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 799 Arthur C. Ouwehand . Kirsti Tiihonen . Sampo Lahtinen
78
Probiotics in Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 811 Benedetta Cinque . Paola Palumbo . Cristina La Torre . Esterina Melchiorre . Daniele Corridoni . Gianfranca Miconi . Luisa Di Marzio . Maria Grazia Cifone . Maurizio Giuliani
Part 4
Toxicological/Safety and General Considerations . . . . . . . . . . . . . . . . . . . . 821
Safety Evaluation for the Elderly Population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 823 79
Irritant Contact Dermatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 825 Florian Seyfarth . Peter Elsner
80
Susceptibility to Irritation in the Elderly: New Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833 Miranda A. Farage . Kenneth W. Miller . G. Frank Gerberick . Cindy A. Ryan . Howard I. Maibach
81
Safety Evaluation in the Elderly via Dermatological Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . 845 Mario Bramante
82
Dermal Safety Evaluation: Use of Disposable Diaper Products in the Elderly . . . . . . . . . . . . . . . 857 Prashant Rai . Daniel S. Marsman . Susan P. Felter
Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869 83
Aging Skin Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 871 Duane L. Charbonneau . Yen L. Song . Cheng Xu Liu
84
The Vaginal Microbiota in Menopause . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 883 Miranda A. Farage . Kenneth W. Miller . Jack D. Sobel
Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 895 85
Impaired Wound Repair and Delayed Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 897 Matthew J. Ranzer . Luisa A. DiPietro
Wrinkles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 909 86
Facial Wrinkling: The Marquee Clinical Sign of Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 911 Greg G. Hillebrand
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Table of Contents
Scales and Typing System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 919 87
Assessing Quality of Ordinal Scales Depicting Skin Aging Severity . . . . . . . . . . . . . . . . . . . . . . 921 Fabien Valet . Khaled Ezzedine . Denis Malvy . Jean-Yves Mary . Christiane Guinot
88
The Baumann Skin Typing System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 929 Leslie S. Baumann
Part 5
Global Skin Aging and its Management: Perception, Needs, Differences and Responses to Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . 945
Psychosocial Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 947 89
Psychological and Social Implications of Aging Skin: Normal Aging and the Effects of Cutaneous Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 949 Miranda A. Farage . Kenneth W. Miller . Enzo Berardesca . Howard I. Maibach
90
Aging Skin: Some Psychosomatic Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 959 Madhulika A. Gupta
Aging Perception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971 91
Facial Skin Attributes and Age Perception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973 Alex Nkengne . Georgios Stamatas . Christiane Bertin
Gender, Ethnicity and Lifestyle Differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 981 92
Determinants in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983 Miranda A. Farage . Kenneth W. Miller . Howard I. Maibach
93
Gender Differences in Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 999 Sarah Fitzmaurice . Howard I. Maibach
94
Aging in Asian Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1019 Low Chai Ling
Sensitive Skin and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1025 95
Perceptions of Sensitive Skin with Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1027 Miranda A. Farage
96
Aging and Skin Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1047 Michael K. Robinson
Ingredients and Products for Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1053 97
Aging and Anti-aging Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1055 Giuseppina Candore . Giovanni Scapagnini . Calogero Caruso
Table of Contents
98
Cosmetics and Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1063 Robert L. Bronaugh . Linda M. Katz
99
Cosmetic Anti-aging Ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1069 Donald L. Bissett . Mary B. Johnson
100
Topical Growth Factors for Skin Rejuvenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079 Vijayeta Rangarajan . Frank Dreher
101
Topical Peptides and Proteins for Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1089 Farzam Gorouhi . Howard I. Maibach
Fem Care Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1119 102
Solutions and Products for Managing Female Urinary Incontinence . . . . . . . . . . . . . . . . . . . . . 1121 David J. Caracci
103
Changes in Vulvar Physiology and Skin Disorders with Age and Benefits of Feminine Wipes in Postmenopausal Women . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1127 Miranda A. Farage . Kenneth W. Miller . William J. Ledger
Cosmetic Surgeries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1137 104
A New Paradigm for the Aging Face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1139 Samuel M. Lam
105
Cosmetic Surgery in the Elderly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1147 Dwight Scarborough . Kimberly M. Eickhorst . Emil Bisaccia
106
Facial Rejuvenation: A Chronology of Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175 Alexander S. Donath
Part 6 107
Global Market Place for the Aged . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185
Marketing and Product Design of Anti-aging Skin Care Products . . . . . . . . . . . . . . . . . . . . . . . 1187 Nancy C. Dawes
108
Key Trends Driving Anti-aging Skin Care in 2009 and Beyond . . . . . . . . . . . . . . . . . . . . . . . . . 1197 Mary Carmen Gasco-Buisson
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1207
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Editors
Miranda A. Farage, Ph.D. Principal Scientist The Procter & Gamble Company 6110 Center Hill Avenue Cincinnati, OH 45224 USA
[email protected] Kenneth W. Miller, Ph.D. Associate Director The Procter & Gamble Company 6110 Center Hill Avenue Cincinnati, OH 45224 USA
[email protected]
Howard I. Maibach, M.D. Professor Department of Dermatology University of California, School of Medicine San Francisco, CA 94122 USA
[email protected]
Contributors
Ossama Abbas Department of Dermatology American University of Beirut Medical Center Beirut Lebanon Jean Adamus Unilever R&D Trumbull, CT USA Mohamed A. Adly Department of Zoology Faculty of Science Sohag University Sohag Egypt Denize Ainbinder School of Pharmacy Faculty of Medicine The Hebrew University of Jerusalem Jerusalem Israel Ali Alikhan Department of Dermatology School of Medicine University of California, Davis Sacramento, CA USA Fatima S. Alikhan University of California, Berkeley Berkeley, CA USA Satoshi Amano Shiseido Research Center Tsuzuki-ku, Yokohama Japan
Melina C. Armenaka Department of Dermatology University of Athens Kesariani, Athens Greece Jorge Arrese-Estrada Laboratory of Skin Bioengineering and Imaging Department of Dermatopathology University Hospital of Lie`ge Lie`ge Belgium Hanan Assaf Department of Dermatology and Venereology Faculty of Science Sohag University Sohag Egypt Daniel Asselineau L’Ore´al Life Sciences Research Clichy France Gurinder Singh Bains Department of Physical Therapy School of Allied Health Professions Loma Linda University Loma Linda, CA USA Leslie S. Baumann Department of Dermatology and Baumann Cosmetic Medicine and Research Institute University of Miami Miami, FL USA Thomas Blatt Skin Research Centre Beiersdorf AG Hamburg Germany
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Contributors
Enzo Berardesca San Gallicano Dermatological Institute IRRCS Roma Rome Italy Christiane Bertin Johnson & Johnson Consumer France Issy-les-Moulineaux France Marianne Berwick Division of Epidemiology and Biostatistics Department of Internal Medicine University of New Mexico Albuquerque, NM USA Tapan K. Bhattacharyya Department of Otolaryngology-Head & Neck Surgery University of Illinois, Chicago Chicago, IL USA Emil Bisaccia College of Physicians and Surgeons Columbia University New York, NY USA Donald L. Bissett The Procter & Gamble Company Cincinnati, OH USA Donald L. Bjerke The Procter & Gamble Company Cincinnati, OH USA Carol Bosko Unilever R&D Trumbull, CT USA Mario Bramante Procter & Gamble Service GmbH Schwalbach am Taunus, Hesse Germany
Douglas E. Brash Departments of Therapeutic Radiology, Genetics and Dermatology School of Medicine Yale University New Haven, CT USA Robert L. Bronaugh CFSAN Food and Drug Administration College Park, MD USA Ste´phane Bre´zillon Laboratory of Biochemistry and Molecular Biology Faculty of Medicine University of Reims Champagne-Ardenne Reims France Giuseppina Candore Department of Pathobiology and Biomedical Methodologies University of Palermo Palermo Italy David J. Caracci The Procter & Gamble Company Cincinnati, OH USA Calogero Caruso Department of Pathobiology and Biomedical Methodologies University of Palermo Palermo Italy Duane L. Charbonneau The Procter and Gamble Company Cincinnati, OH USA Alexandra Charruyer Department of Dermatology and Institute for Regeneration Medicine University of California, San Francisco San Francisco, CA USA
Contributors
Maria Grazia Cifone Department of General Pathology Faculty of Medicine and Surgery University of L’Aquila Coppito Italy Benedetta Cinque Department of Health Sciences University of L’Aquila Coppito Italy Gae¨lle Claviez-Homberg L’Ore´al Life Sciences Research Clichy France Daniele Corridoni Department of Health Sciences University of L’Aquila Coppito Italy Jonathan M. Crowther Procter & Gamble Technical Centres Ltd Egham, Surrey UK Akira Date Procter & Gamble Japan K.K Kobe, Hyogo Japan Nancy C. Dawes The Procter and Gamble Company Cincinnati, OH USA Luisa A. DiPietro Center for Wound Healing and Tissue Regeneration College of Dentistry University of Illinois, Chicago Chicago, IL USA Alexander S. Donath Cincinnati Facial Plastic Surgery Cincinnati, OH USA
Frank Dreher NEOCUTIS Inc. San Francisco, CA USA Kimberly M. Eickhorst Procedural Dermatology Fellow Morristown, NJ USA Peter Elsner Department of Dermatology and Allergology University of Jena Jena Germany Alex Eshaghian Department of Internal Medicine University of New Mexico Albuquerque, NM USA Khaled Ezzedine Department of Dermatology CHU Saint-Andre´ Bordeaux France Miranda A. Farage The Procter & Gamble Company Cincinnati, OH USA Susan P. Felter The Procter & Gamble Company Cincinnati, OH USA Sarah Fitzmaurice School of Medicine University of California, Davis Sacramento, CA USA Sara Flores Department of Dermatology School of Medicine University of California, San Francisco San Francisco, CA USA
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Contributors
Joachim Fluhr Bioskin, GmBH Hamburg Germany G. Frank Gerberick The Procter & Gamble Company Cincinnati, OH USA Mary Carmen Gasco-Buisson The Procter and Gamble Company Cincinnati, OH USA Paolo U. Giacomoni Clinique Laboratories Melville, NY USA Ruby Ghadially Department of Dermatology University of California, San Francisco and Veteran’s Affairs Medical Center San Francisco, CA USA Sarah Girardeau L’Ore´al Life Sciences Research Clichy France Maurizio Giuliani Department of Health Sciences University of L’Aquila Coppito Italy Francesca Giusti Department of Dermatology University of Modena and Reggio Emilia Modena Italy Farzam Gorouhi Department of Dermatology University of California, San Francisco San Francisco, CA USA
Christiane Guinot Biometrics and Epidemiology Unit CE.R.I.E.S. Neuilly-sur-Seine France Madhulika A. Gupta Department of Psychiatry Schulich School of Medicine and Dentistry University of Western Ontario London, ON Canada Bahman Guyuron Department of Plastic Surgery University Hospitals of Cleveland and Case Western Reserve University Cleveland, OH USA Tomohiro Hakozaki The Procter & Gamble Company Cincinnati, OH USA Stacy S. Hawkins Unilever R&D Trumbull, CT USA Timothy P. Heffernan Departments of Therapeutic Radiology, Genetics and Dermatology School of Medicine Yale University New Haven, CT USA Peter Helmbold Department of Dermatology University of Heidelberg Heidelberg Germany Fre´de´rique Henry Laboratory of Skin Bioengineering and Imaging Department of Dermatopathology University Hospital of Lie`ge Lie`ge Belgium
Contributors
Andia Heydari University of California, Los Angeles Los Angeles, CA USA Panthea Heydari University of California, Los Angeles Los Angeles, CA USA Greg G. Hillebrand The Procter & Gamble Company Cincinnati, OH USA Tetsuji Hirao Shiseido Research Center Tsuzuki-ku, Yokohama Japan Regina Hourigan Colgate-Palmolive Company Piscataway, NJ USA Michael F. Hughes Office of Research and Development US Environmental Protection Agency National Health and Environmental Effects Research Laboratory Research Triangle Park, NC USA Young Hui University of California, San Francisco San Francisco, CA USA
Mary B. Johnson The Procter & Gamble Company Cincinnati, OH USA Alexandra Katsarou Department of Dermatology University of Athens Kesariani, Athens Greece Linda M. Katz Office of Cosmetics and Colors CFSAN Food and Drug Administration College Park, MD USA Abdul Ghani Kibbi Department of Dermatology American University of Beirut Medical Center Beirut Lebanon Won-Serk Kim Department of Dermatology Kangbuk Samsung Hospital Sungkyunkwan University School of Medicine Seoul Korea Jean Krutmann Environmental Health Research Institute (IUF) Heinrich-Heine-University Duesseldorf Germany
Mahmoud R. Hussein Department of Pathology Faculty of Medicine Assuit University Abha Saudi Arabia
Cristina La Torre Department of Health Sciences University of L’Aquila Coppito Italy
Qunshan Jia The Procter & Gamble Company Cincinnati, OH USA
Sampo Lahtinen Danisco Health and Nutrition Kantvik Finland
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Contributors
Samuel M. Lam Willow Bend Wellness Center and Lam Facial Plastic Surgery Center & Hair Restoration Institute Plano, TX USA
Denis Malvy Department of Internal Medicine and Infectious Diseases CHU St.Andre´ Bordeaux France
William J. Ledger Department of Obstetrics and Gynecology Cornell University-Weill Medical College New York, NY USA
Franc¸ois-Xavier Maquart Laboratoire Central de Biochimie Hoˆpital Robert Debre´ Reims France
Jacquelyn Levin Arizona College of Osteopathic Medicine Midwestern University Glendale, AZ USA
Slaheddine Marrakchi Department of Dermatology Hedi Chaker Hospital Sfax Tunisia
Davina A. Lewis Department of Dermatology School of Medicine Indiana University Indianapolis, IN USA
Daniel S. Marsman The Procter & Gamble Company Cincinnati, OH USA
Low Chai Ling The Sloane Clinic Singapore Singapore Cheng Xu Liu The Procter and Gamble Company Cincinnati, OH USA Howard I. Maibach Department of Dermatology School of Medicine University of California, San Francisco San Francisco, CA USA Evgenia Makrantonaki Departments of Dermatology, Venereology Allergology and Immunology Dessau Medical Center Dessau Germany
Jean-Yves Mary INSERM Department of Biostatistics and Clinical Epidemiology Saint-Louis Hospital Paris France Luisa Di Marzio Department of Health Sciences University of L’Aquila Coppito Italy Paul J. Matts Procter & Gamble Technical Centres Ltd Egham, Surrey UK Esterina Melchiorre Department of Health Sciences University of L’Aquila Coppito Italy
Contributors
Helen Meldrum Unilever R&D Trumbull, CT USA Joseph Merregaert Laboratory of Molecular Biotechnology Department of Biomedical Sciences University of Antwerp Antwerp Belgium Gianfranca Miconi Department of Health Sciences University of L’Aquila Coppito Italy Kenneth W. Miller The Procter & Gamble Company Cincinnati, OH USA Sole`ne Mine L’Ore´al Life Sciences Research Clichy France Akimichi Morita Department of Geriatric and Environmental Dermatology Nagoya City University Nagoya Japan Kouichi Nakagawa RI Research center Fukushima Medical University Fukushima Japan Flore Nallet L’Ore´al Life Sciences Research Clichy France J. Frank Nash The Procter & Gamble Company Cincinnati, OH USA
Isaac M. Neuhaus Department of Dermatology University of California, San Francisco San Francisco, CA USA Paul Nghiem Departments of Therapeutic Radiology, Genetics and Dermatology School of Medicine Yale University New Haven, CT USA
John Nip Unilever R&D Trumbull, CT USA Alex Nkengne Johnson & Johnson Consumer France Issy-les-Moulineaux France Kimberly G. Norman Vanderbilt University Medical Center Nashville, TN USA Mutsumi Okazaki Department of Plastic and Reconstructive Surgery Tokyo Medical and Dental University Bunkyo-ku, Tokyo Japan
Arthur C. Ouwehand Danisco Health and Nutrition Kantvik Finland
Noritaka Oyama Department of Dermatology School of Medicine Fukushima Medical University Fukushima Japan
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Contributors
Herve´ Pageon L’Ore´al Life Sciences Research Clichy France Paola Palumbo Department of Health Sciences University of L’Aquila Coppito Italy Philippe Paquet Laboratory of Skin Bioengineering and Imaging Department of Dermatopathology University Hospital of Lie`ge Lie`ge Belgium Byung-Soon Park Department of Dermatology College of Medicine Seoul National University Seoul Korea Jerrold Scott Petrofsky School of Allied Health Loma Linda University Loma Linda, CA USA Ge´rald E. Pie´rard Laboratory of Skin Bioengineering and Imaging Department of Dermatopathology University Hospital of Lie`ge Lie`ge Belgium Claudine Pie´rard-Franchimont Laboratory of Skin Bioengineering and Imaging Department of Dermatopathology University Hospital of Lie`ge Lie`ge Belgium S. Brian Potterf Unilever R&D Trumbull, CT USA
Rupa Pugashetti UCSF Psoriasis Treatment Center School of Medicine University of California, Irvine Irvine, CA USA Pascale Quatresooz Laboratory of Skin Bioengineering and Imaging Department of Dermatopathology University Hospital of Lie`ge Lie`ge Belgium Prashant Rai Procter and Gamble Japan KK Higashinada-ku Japan Vijayeta Rangarajan NEOCUTIS Inc. San Francisco, CA USA Matthew J. Ranzer Center for Wound Healing and Tissue Regeneration College of Dentistry University of Illinois, Chicago Chicago, IL USA Christina Raschke Department of Dermatology and Allergology University of Jena Jena Germany Suresh I. S. Rattan Laboratory of Cellular Ageing Department of Molecular Biology Aarhus University Aarhus Denmark Anthony V. Rawlings AVR Consulting Ltd Northwich, Cheshire England UK
Contributors
Glen Rein Estee Lauder Companies Melville, NY USA Sylvie Ricois L’Ore´al Life Sciences Research Clichy France Michael K. Robinson The Procter & Gamble Company Cincinnati, OH USA Sheila Rocha Unilever R&D Trumbull, CT USA David J. Rowe University Hospitals Case Western Reserve University Cleveland, OH USA Nelly Rubeiz Department of Dermatology American University of Beirut Medical Center Beirut Lebanon Cindy A. Ryan The Procter & Gamble Company Cincinnati, OH USA Shingo Sakai Basic Research Laboratory Kanebo Cosmetics Inc. Kanagawa Japan Salah Salman Department of Dermatology American University of Beirut Medical Center Beirut Lebanon
Giovanni Scapagnini Department of Health Sciences University of Molise Campobasso Italy Dwight Scarborough Division of Dermatology Ohio State University Hospital Columbus, OH USA Peter Schroeder Environmental Health Research Institute (IUF) Heinrich-Heine-University Duesseldorf Germany Stefania Seidenari Department of Dermatology University of Modena and Reggio Emilia Modena Italy Sandy Sercu Laboratory of Molecular Biotechnology Department of Biomedical Sciences University of Antwerp Antwerp Belgium Florian Seyfarth Department of Dermatology and Allergology University of Jena Jena Germany Susan N. Sherman SNS Research Cincinnati, OH USA William Shingleton Unilever R&D Colworth UK James E. Sligh Skin Diseases Research Center Vanderbilt University Medical Center Nashville, TN USA
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Contributors
Jack D. Sobel Division of Infectious Diseases Department of Internal Medicine Wayne State University Detroit, MI USA Yen L. Song The Procter and Gamble Company Cincinnati, OH USA Dan F. Spandau Department of Dermatology School of Medicine Indiana University Indianapolis, IN USA Georgios Stamatas Johnson & Johnson Consumer France Issy-les-Moulineaux France Robert Stern Department of Pathology Touro College of Osteopathic Medicine New York, NY USA Paul R. Summers University Health Care University of Utah Salt Lake City, UT USA Cheri L. Swanson The Procter & Gamble Company Cincinnati, OH USA Hachiro Tagami Department of Dermatology Tohoku University School of Medicine Sendai Japan Se´verine Teluob L’Ore´al Life Sciences Research Clichy ce´dex France
Zack Thompson University of California, San Francisco San Francisco, CA USA Haw-Yueh Thong Department of Dermatology University of California, San Francisco San Francisco, CA USA Kirsti Tiihonen Danisco Health and Nutrition Kantvik Finland Salina M. Torres Division of Epidemiology and Biostatistics Department of Internal Medicine University of New Mexico Albuquerque, NM USA Elka Touitou School of Pharmacy Faculty of Medicine The Hebrew University of Jerusalem Jerusalem Israel Jeffrey B. Travers Department of Dermatology School of Medicine Indiana University Indianapolis, IN USA Joel Tsevat Department of Internal Medicine College of Medicine University of Cincinnati Cincinnati, OH USA Giuseppe Valacchi Department of Biomedical Sciences University of Siena Siena Italy
Contributors
Fabien Valet INSERM, Biostatistics and Clinical Epidemiology DBIM, Saint-Louis Hospital University Paris 7 France
Klaus-Peter Wittern Skin Research Centre Beiersdorf AG Hamburg Germany
Shilpa Vora Unilever R&D Bangalore India
Emmanuelle Xhauflaire-Uhoda Laboratory of Skin Bioengineering and Imaging Department of Dermatopathology University Hospital of Lie`ge Lie`ge Belgium
Yanusz Wegrowski Laboratory of Biochemistry and Molecular Biology Faculty of Medicie University of Reims Champagne-Ardenne Reims France Horst Wenck Skin Research Centre Beiersdorf AG Hamburg Germany Klaus-Peter Wilhelm proDERM Institut fuer Angewandte Dermatologische Forschung GmbH Hamburg Germany
Daniel B. Yarosh Estee Lauder Companies Melville, NY USA Christos C. Zouboulis Institute of Clinical Pharmacology & Department of Toxicology Charite´ Universitaetsmedizin Berlin Campus Benjamin Franklin Berlin Germany He´le`ne Zucchi L’Ore´al Life Sciences Research Clichy France
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20 Adipose-derived Stem Cells and their Secretory Factors for Skin Aging Byung-Soon Park . Won-Serk Kim
Introduction The term ‘‘stem cell’’ has attracted increasing attention of the scientific community as well as of the general public. In many aspects, however, it is still confusing and difficult to understand and interpret information about stem cells. They are vital to humans for numerous reasons. Groups of stem cells in some adult tissues give rise to replacement cells for the tissues that are destroyed through injury, disease, or aging [1]. Knowledge relating to how healthy cells replace diseased or otherwise damaged cells, would allow development of medical therapies focusing on creation of compatible cell lines to replace aged or diseased cells in the body. The concept of regenerative medicine using the body’s own stem cells and growth factors to repair tissue may be realizable as science and clinical experience converge to develop alternative therapeutic strategies to treat the damaged or diseased tissue. Stem cell-based therapies are also being tried in tissue engineering: The aim of tissue engineering is to repair and regenerate damaged organs or tissues using a combination of cells, biomaterials, and cytokines [1–4]. This chapter addresses the human subcutaneous adipose tissue as a promising source of adult stem cells. Adipose-derived stem cells (ADSCs) may offer a solution for the problem of limited availability of human cells that are capable of self-renewal and differentiation. ADSCs can be easily obtained from liposuction of human adipose tissue, cultured in a large scale, and display multi-lineage developmental plasticity. In addition, ADSCs secrete various cytokines and growth factors, which control and manage the damaged neighboring cells, and this has been identified as essential functions of ADSCs [5–7]. As reviewed elsewhere in this book, aging and photoaging are complex processes involving the wound-healing cascade and/or repetitive oxidative stress. Conventional anti-aging skin treatments such as light-based or radiofrequency devices and/or peelings have been less than satisfactory because their primary mechanism is mainly inducing new collagen synthesis via activation of dermal fibroblasts. On the basis of previous studies that
demonstrated wound healing, antioxidant, antiwrinkle, and antimelanogenic effects of ADSCs and their secretory factors, they may be good candidates for the treatment of aging [5–9]. Therefore, this chapter describes the authors’ recent research and clinical developments on the anti-aging effects of ADSCs and their secretory factors.
Stem Cells and ADSCs Stem cells are a population of immature tissue precursor cells capable of self-renewal and provision of multi-lineage differentiable cells for tissues. Although embryonic stem cell has multi-potency, there are many limitations such as difficulties in control of differentiation and issues relating to ethics. As a result, use of adult stem cells with fewer implicating issues is becoming an area of increased interest in stem cell medicine. Given the vast potential of treatments utilizing stem cells, validation and evaluation regarding safety and efficacy will result in greater benefits.
ADSCs and Regeneration Due to the lack of a specific and universal molecular marker for adult stem cells, functional assays for multiple differentiations must be used to identify stem cells in a tissue. Mesenchymal stem cells (MSCs) were first characterized in bone marrow, but many studies have reported the existence of MSCs in the connective tissue of several organs [10, 11]. The role of these cells is not entirely clear, but they are generally believed to constitute a reserve for tissue maintenance and repair. It was recently demonstrated that the most abundant and accessible source of adult stem cells is adipose tissue. The yield of MSCs from adipose tissue is approximately 40-fold greater than that from bone marrow [12–14]. The following are the highly consistent, although not identical, expression profiles of cell-surface proteins on ADSCs [2, 15]: adhesion molecules, receptor molecules, surface enzymes, extracellular matrix (ECM) proteins,
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_20, # Springer-Verlag Berlin Heidelberg 2010
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and glycoproteins. However, hematopoietic cell markers such as CD14, CD31, and CD45 are not expressed. Interestingly, the immunophenotype of ADSCs resembles that reported for other adult stem cells prepared from human bone marrow (bone marrow stromal cell [BMSC]) and skeletal muscle [2]. Differentiation of ADSCs is not restricted to the adipocyte lineage, but they can be differentiated into chondrocyte, osteocyte, cardiomyocyte, neuron, etc. [16, 17]. In addition, activity comparison with BMSC revealed a similar regenerative capacity. Therefore, this abundant and accessible cell population has potential clinical utility for regenerating damaged or aged tissue and tissue engineering. As with many rapidly developing fields, diverse names have been proposed to describe the plastic-adherent cell population isolated from collagenase digests of adipose tissue: adipose-derived stem/stromal cells, adiposederived adult stem cells, adipose-derived adult stromal cells, adipose stromal cells (ASCs), adipose mesenchymal stem cells (AdMSCs), lipoblast, pericyte, preadipocyte, and processed lipoaspirate (PLA) cells. To address the confusion due to diverse nomenclature, the International Fat Applied Technology Society reached a consensus to adopt the term ‘‘adipose-derived stem cells’’ to identify the isolated, plastic-adherent, multipotent cell population. Questioning the validity of the term ‘‘stem cell’’, led to the use of the acronym to mean ‘‘adipose-derived stromal cells’’ [18]. Although studies are limited, the quality and quantity of the ADSCs varies according to interperson differences, the harvest site, harvesting method, and culture conditions. Age and sex are the most obvious of the interperson differences. Stem cell recovery varies between subcutaneous white adipose tissue depots [19, 20]. Yield and growth characteristics of ADSC (> Fig. 20.1) are also affected by the type of surgical procedure used for adipose tissue harvesting. Resection and tumescent liposuction seem to be preferable above ultrasound-assisted liposuction [21].
Mechanism of Action for Regeneration Stem cell therapy is a safe, practical, and effective source for repair of damaged tissue [22, 23]. Despite rapid translation to the bedside, the mechanism of action for regeneration is not well characterized. It was initially hypothesized that immature stem cells migrate to the injured area, differentiate into the phenotype of injured
. Figure 20.1 ADSCs display adherent and fibroblastic morphology. They show abundant endoplasmic reticulum and large nucleus relative to the cytoplasmic volume (Reprinted with permission from Elsevier, Kim WS et al. [6])
tissue, repopulate the diseased organ with healthy cells, and subsequently repair the tissue (building-block function). However, this theory has some drawbacks because the levels of engraftment and survival of engrafted cells are too low to be therapeutically relevant [24]. In addition, acute stem cell-mediated improvement within days or even hours makes it difficult to fully explain the mechanisms by which regeneration occurs [25, 26]. Instead, much of the functional improvement and attenuation of injury afforded by stem cells can be repeated by treatment with cell-free conditioned media derived from ADSCs (ADSCCM) [27]. Thus, it can be deduced that ADSCs may exert their beneficial effects via complex paracrine actions (manager function) in addition to building-block function.
Adipose-derived Stem Cells and their Secretory Factors for Skin Aging
Proteomic Analysis of ADSCs and Their Secretomes Proteomics, large-scale studies of proteins, can be used to analyze the intracellular and secretory proteins of ADSCs. For example, Roche et al., conducted a 2-DE gel analysis of BMSCs and ADSCs, and confirmed the similarity [28]. Zvonic et al., also analyzed the ADSC-CM by 2-DE gel electrophoresis, detected approximately 300 features from ADSC-CM, and found that secretomes are up-/downregulated by induction of adipogenesis [29]. Although the intracellular and secretory proteins of ADSCs have been analyzed through 2-DE-coupled mass spectrometry or non-gel-based mass spectrometry, the active proteins of ADSCs responsible for the tissue regeneration are not fully identified. This may be due to the fact that studies using proteomics has limitations as this approach is capable of analyzing highly abundant proteins only. Therefore, new mass spectrometry-based proteomic analysis techniques for stem cell proteins in correlation with other state-of-the-art analytical tools and functional study by neutralizing the candidate proteins are needed to clearly characterize the active proteins of regeneration.
Diverse Pharmacologic Actions of ADSCs and Their Secretory Factors Wound-Healing Effect of ADSCs Several studies of the pathophysiology of photoaging have detected similarities with certain aspects of acute and/or chronic wounds. Histologically, photoaged skin shows marked alterations in ECM composition. Skin wound repair by adult stem cells was originally demonstrated using BMSC. Wu et al. showed that BMSC injection around the wound significantly enhanced wound healing in normal and diabetic mice compared with that of allogeneic neonatal dermal fibroblasts [30]. Sasaki et al. demonstrated that BMSCs can differentiate into multiple skin cell types including keratinocytes, pericytes, and endothelial cells, which contribute to wound repair [31]. Notably, analyses of proteins in conditioned medium of BMSC (BMSC-CM) indicated that BMSCs secret distinctively different cytokines and chemokines compared to dermal fibroblasts [32]. ADSCs have surface markers and gene profiling similar to BMSCs and their soluble factors are not significantly different [6, 10]. Given their convenient isolation compared with BMSCs and extensive proliferative capacities ex vivo, ADSCs hold great promise
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for use in wound repair and regeneration. However, there is little evidence demonstrating the wound-healing effects of ADSCs. It was also demonstrated that ADSCs accelerate wound healing, especially with regard to fibroblast activation [6]. They promote proliferation of dermal fibroblasts, not only by direct cell-to-cell contact, but also by paracrine activation through secretory factors. This fibroblast-stimulating effect of ADSCs was superior to that of the fibroblasts. Furthermore, ADSC-CM enhanced secretion of type I collagen from dermal fibroblasts and stimulated fibroblast migration in in vitro wound-healing models. ADSCs secreted a variety of growth factors such as basic fibroblast growth factor (bFGF), KGF, TGF-b, hepatocyte growth factor (HGF), and VEGF into the conditioned medium, which might mediate the woundhealing effect of ADSCs. In addition to the in vitro evidence, the wound-healing effect of ADSCs was also verified in an animal study, which showed that topical administration of ADSCs significantly reduced the wound size (34% reduction) and accelerated the re-epithelialization at the wound edge (> Fig. 20.2). Similar to ADSC treatment, ADSC-CM treatment also accelerated wound healing in laser-induced burn mouse models (authors’ unpublished data). In this experiment, burn wounds were made by laser surgery in the epidermis and they were significantly reduced by single and multiple administration of ADSC-CM. As ADSCs are physiologically located beneath dermal fibroblasts, they may interact with dermal fibroblasts. However, ADSCs and secretomes of ADSCs may reach the epidermis in wounded area and may affect the recovery of this layer. As such, ADSC-CM was treated in cultured primary human keratinocytes and shown to increase the proliferation and migration of keratinocytes (authors’ unpublished data). This result suggests that secretomes of ADSC also accelerate the healing of epidermal layer.
Antioxidant and Antimelanogenic Effects of ADSC Reactive oxygen species (ROS) produced in the catalytic reactions by many environmental stimuli may be involved in the pathogenesis of a number of skin disorders including photoaging, photosensitivity diseases, and some types of cutaneous malignancy. Antioxidants, as a popular term in drug and cosmetics, take the form of enzymes, hormones, vitamins, and minerals. In biological systems, the normal processes of oxidation produce highly reactive free radicals, which may continue to damage even the
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. Figure 20.2 Wound healing effect of ADSCs in nude mice. Artificial wounds were made using a 6-mm punch biopsy and ADSCs were topically applied. The wound size was reduced significantly in the ADSC-treated side (right side of the back) 7 days after surgery (Reprinted with permission from Elsevier, Kim WS et al. [6])
body’s own cells. Antioxidants scavenge free radicals before they get a chance to harm the body. As of now, there are few reports on the antioxidant action of stem cells. However, some evidences support the protective role of secretomes of ADSCs against the skin damage induced by reactive oxygen species. For example, IGF reportedly protects fibroblasts and intestinal epithelial cells from free radicals [33, 34]. HGF protects the retinal pigment epithelium against oxidative stress induced by glutathione depletion [35]. Pigment epithelium-derived factor (PEDF) is an anti-angiogenic/neurotropic factor and has been shown to have antioxidant effects [36]. Interleukin-6 (IL-6) reduces the epithelial cell death induced by hydrogen peroxide [37]. In addition, subtypes of superoxide dismutase (SOD) are expressed and secreted from ADSC [38]. Therefore, antioxidant function of ADSC was investigated in dermal fibroblasts after inducing chemical oxidative stress by the tert-butyl hydroperoxide (tbOOH). Morphological change and cell survival assay revealed that incubation with ADSC-CM aided dermal fibroblasts to resist free radicals induced by tbOOH. In addition, activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were enhanced in the dermal fibroblasts treated with ADSC-CM. In a cell cycle analysis, ADSC-CM treatment reversed the apoptotic cell death induced by ROS, which was demonstrated by a significant decrease of subG1 phase of dermal fibroblasts [8]. Photoaging is believed to be responsible for up to almost 80% of the skin changes commonly attributed to the aging process. The study further investigated the antioxidant and protective effects of ADSCs in the photodamage of the primarily cultured dermal fibroblasts (> Fig. 20.3). In this experiment, ADSC-CM pretreatment significantly
. Figure 20.3 Antioxidant effect of ADSCs in UVB-irradiated fibroblasts as shown by cell cycle analysis of DNA contents. Untreated fibroblasts showed little or no sub-G1 phases (a). However, UVB irradiation significantly increased sub-G1 (apoptotic) cells (b), which were reversed by ADSC-CM pretreatment (c) (Reprinted with permission from Elsevier, Kim WS et al. [5])
Adipose-derived Stem Cells and their Secretory Factors for Skin Aging
reduced the apoptosis of dermal fibroblasts from UVBinduced damage, which was demonstrated by a significant decrease of sub-G1 phase of dermal fibroblasts after ADSC-CM pretreatment. In addition, ADSC-CM treatment increased the production of collagen and reduced the expression of matrix metalloproteinase-1 in the dermal fibroblasts. These results indicated that ADSCs can play a key role in protecting dermal fibroblast from UVBinduced oxidative stress [5]. As antioxidants inhibit the chemical reactions leading to melanin formation, change the type of melanin formed,
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and interfere with the distribution of pigment and melanosome transfer, they are good candidates for skin whitening resources. As ADSC-CM is a free radical scavenger and has potent antioxidant activity, antimelanogenic effect of ADSC was investigated. ADSC-CM treatment inhibited the synthesis of melanin and the activity of tyrosinase in melanoma B16 cells. In addition, expressions of tyrosinase and tyrosinase-relating protein 1 were down-regulated by ADSC-CM treatment, which indicated the mechanism of action for antimelanogenic effect of ADSCs and their soluble factors (> Fig. 20.4) [9].
. Figure 20.4 (a) Antimelanogenic effect of ADSC-CM. Expression of MITF and TRP2 remained unchanged, but expressions of tyrosinase and TRP1 were down-regulated by ADSC-CM treatment in B16 melanoma cells. (b) The inhibitory effect of ADSC on melanin synthesis is schematically represented (Reproduced with permission from Pharmacological Society of Japan, Kim WS et al. [9])
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Animal Studies for Skin Aging To study the effects on in vivo skin, ADSCs (1 106 cells) and ADSCs-CM were intradermally injected on the back of a micropig, twice in a 14-day interval (n = 3). One month after the second injection, skin samples were obtained at the treatment and the control sites of adjacent normal skin. Although the increase in the dermal thickness was not significant, increased collagen expression was noted by western blot in the ADSCs- and ADSCs-CMtreated skin samples (> Fig. 20.5) [7]. In another experiment, photodamage was induced by an 8-week UVB irradiation in hairless mice. The irradiation dose was one MED (minimal erythema dose; 60 mJ/cm2) in the first 2 weeks, two MED in the third week, three MED in the forth week, and four MED in the fifth through eighth weeks. After wrinkle induction, varying numbers of ADSCs (A group: control; B group: 1 103 cells; C group: 1 104 cells; and D group: 1 105 cells) were subcutaneously injected into the mice (n = 8 for each group). In a replica analysis, parameters involving skin roughness were improved with mid-level and higher dose groups of ADSCs (C and D group) (> Fig. 20.6). Dermal thickness was increased in the ADSC-injected groups (16% and 28% in C and D groups, respectively) and ECM contents in the dermis were also increased by Massson’s trichrome staining results of collagen (blue) in the ADSCtreated groups (> Fig. 20.7). As cell transplantation between species mediates immune rejection, the survival of
ADSC from humans was investigated after injection of ADSCs labeled with PKH26 (red color, > Fig. 20.8 inset). As shown in > Fig. 20.8, survival of the ADSCs was clearly demonstrated [5].
Clinical Application of ADSCs and the ADSC Protein Extract ADSCs and the ADSC Protein Extract for Skin Aging As a pilot study, intradermal injections of purified autologous PLA cells (1 106 cells), which contain approximately 20–30% ADSCs, were tried with photoaged skin of one patient [7] after informed conset. The female patient had two successive injections at 2-week intervals. Two months after the second injection, the patient showed improvements in general skin texture and wrinkling as evidenced by medical photographs of periorbital wrinkles. Measurements of dermal thickness by a 20 MHz high-frequency ultrasonographs (Dermascan-C, Cortex, Hadsund, Denmark) also indicated increased thickness (2.054 vs. 2.317 mm) (> Fig. 20.9). In a large-scale pilot study, the effects of the ADSC protein extract applied transdermally in the treatment of the various signs of skin aging were evaluated: (1) wrinkles, (2) acquired pigmentary lesions, and (3) dilated pores [39]. Korean patients visiting for the treatment of
. Figure 20.5 Micropig experiment shows the change of dermal thickness without (a) and with (b) intradermal injections of ADSCs. Increased collagen expression was noted by western blot in the ADSCs- and ADSCs-CM-treated skin (c) (Reproduced with permission from Wiley-Blackwell, Park BS et al. [7])
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. Figure 20.6 Antiwrinkle effects of ADSCs. Photodamage was induced by 8-week UVB irradiation in hairless mice, and ADSCs were intradermally and subcutaneously injected three times. Wrinkles were evaluated by replica analysis. (a) Control; (b) 1 ¥ 103 cells; (c) 1 ¥ 104 cells; (d) 1 ¥ 105 cells (Reprined with permission from Elsevier, Kim WS et al. [5])
. Figure 20.7 Massson’s trichrome staining shows that collagen contents (blue) are significantly increased in the mid-level and higher dose groups of ADSCs (c and d groups in photodamaged hairless mice experiment) (Reprined with permission from Elsevier, Kim WS et al. [5])
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. Figure 20.8 Survival of ADSCs labeled with PKH26 (insert) injected in the skin of hairless mice. Two weeks after injection, mouse skin block was cryosectioned and counterstained with green-fluorescent nucleic acid stain. ADSCs are stained red (Reprined with permission from Elsevier, Kim WS et al. [5])
skin aging were recruited during September 2006–August 2007. The population (n = 235) aged 28–71 years (mean 41 years) had skin phototypes III and IV with mild to moderate photodamage. The advanced ADSC Protein Extract(AAPE; Prostemics Inc., Seoul, Korea) was applied three to twelve times at two-week intervals. The changes were evaluated objectively by photographic documentation and Robo Skin Aanlyzer CS100/VA100 (Inforward Inc., Tokyo, Japan), and subjectively by patient questionnaire. The evaluation score was based upon the following scales: 0 = poor/worsend; 1 = no change/no change; 2 = fair/mild improvement; 3 = good/moderate improvement; and 4 = excellent/marked improvement. As compared to 47.4% showing good to excellent improvement in wrinkle, 63% of the patients were judged to have good to excellent improvement in acquired pigmentary lesions and dilated pores (> Fig. 20.10). Melasma is a multifactorial disorder caused by sun exposure, hormonal imbalance, and genetic predisposition. In many countries including Asia, melasma ranks among the top ten most common skin conditions. Ethnic differences between Asian and other skin types may influence the efficacy and tolerability of melasma treatments.
. Figure 20.9 Clinical study using intradermal injections of purified autologous PLA cells. Medical photographs of periorbital wrinkles were taken before (a) and after (b) treatment, and dermal thickness was measured by ultrasonographs before (c) and after (d) treatment. Improved general skin texture and increase thickness (2.054 vs. 2.317 mm) were evident 2 months after two injections (b and d) (Reprined with permission from Wiley-Blackwell, Park BS et al. [7])
Adipose-derived Stem Cells and their Secretory Factors for Skin Aging
. Figure 20.10 Objective and subjective evaluation of the protein extracts from ADSC-CM in a large-scale (n = 235) pilot study in terms of: (a) wrinkles, (b) acquired pigmentary lesions, and (c) dilated pores. The evaluation score is based upon the following scales: 0 = poor/worsend; 1 = no change/no change; 2 = fair/mild improvement; 3 = good/moderate improvement; and 4 = excellent/marked improvement. As compared to 47.4% showing good to excellent improvement in wrinkle (a), 63% of the patients were judged to have good to excellent improvement in acquired pigmentary lesions (b) and dilated pores (c)
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Recent clinicopathologic studies on melasma show that lesional skin showed more prominent solar elastosis when compared to the normal skin [40]. Moreover, it has been suggested that interactions between the cutaneous vasculature and melanocytes might have an influence on the development of pigmentation [41]. The coexistence of telangiectasia and/or solar elastosis with melasma points out that photodamage is closely linked to the pathogenesis of melasma. The actions of ADSCs in wound healing, antioxidation, antimelanogenic effects and the reversal of photodamage in vitro, and in animal models prompted the clinicians to bring these biologic actions to bedside. The representative cases with marked response in photoaging and melasma were shown in > Fig. 20.11a–d. These clinical results for the past 4 years suggest that the ADSCs and the protein extract are promising rational strategies for melasma and photodamage.
Combination with Other Procedures and Active Transdermal Delivery Various light source and radiofrequency devices have been used for the treatment of skin aging by selectively heating up the collagen in the dermis to stimulate collagen remodeling. In general, both ablative and nonablative techniques lead to new collagen formation. As ADSCs and their secretory factors promote the wound healing by activating dermal fibroblasts, it can be speculated that when combined, ADSCs and the protein extracts might augment the clinical effects beyond the intrinsic fibroblast-stimulatory effect of the various devices. Based upon the previous documentation of woundhealing and antimelanogenic effects of ADSCs, the efficacy of the protein extract of ADSCs in reducing healing time and PIH or erythema was investigated after fractional CO2 laser treatment (MiXto SX1, Lasering, Italy) in a pilot study as prospective, randomized, placebocontrolled, double-blinded, and split-face setting [42]. CO2 fractional treatments have emerged as one of the new technologies in skin rejuvenation. However, comparatively increased incidence of PIH is problematic especially in dark-skinned patients. In this study, Korean patients of Fitzpatrick skin types III and IV (mean age 45.7 years) with facial wrinkles were treated with full-face fractional CO2 laser (Parameter: 8 W, index level 8). All subjects were randomly allocated to split-face application of either the protein extracts of ADSCs or emollient only. Serial photographs were taken at each visit during the treatment and three-month follow-up period. Marked difference in
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. Figure 20.11 The representative cases with marked response before (a, c) and after (b, d) treatment with the protein extracts of ADSCs
the duration of erythema and healing was observed (> Fig. 20.12). The quality of wound healing was noted to be improved. This therapy was well-tolerated by majority of patients with minimal adverse effects. It was concluded that the protein extracts of ADSCs can be safely and effectively used to prevent PIH and to accelerate wound healing after fractional CO2 laser treatment in dark skin. As the secretory factors of ADSC generally contain ingredients of large molecular weights, various new ‘‘active’’ enhancement technologies designed to transiently circumvent the barrier function of the stratum corneum would be required for transdermal delivery: e.g., iontophoresis, sonophoresis, electroporation, or microneedle arrays or skinstamp.
Conclusion The current topics of increasing interest in the dermatological field are anatomical–functional damage to the skin
and every possible means to counteract the injurious effects. In the beginning, ADSCs were shown to increase the survival rate in fat transplantation [43]. This chapter explains that ADSCs and their secretory factors have diverse pharmacologic effects for skin aging. However, clinical application of cultured ADSCs for human skin is in the early stage and might be related to issues/concerns: the threat of passing on viruses, passing on diseases from other animal source nutrients to cultured stem cells in the laboratory, uncontrolled growth, and misdirected growth, especially of embryonic stem cells. In addition, ADSCs have to overcome the obstacles in that they are difficult both to handle and to commercialize in an industrial point of view: how to store the ADSCs, the containers to store them, how to transport them to the point, and the shelf life in various environments. Therefore, new methods and materials to overcome these limitations are needed. Secretomes of ADSC have some advantages over cell-based therapies and might have greater potential in skin regeneration, because they can
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. Figure 20.12 A split-face comparison shows that the application of the protein extract of ADSCs results in less intense erythema and microcrusting 2 days after fractional CO2 laser resurfacing
be manufactured in a large scale with long-term stability and they are relatively devoid of safety issues. As such, the study demonstrated that photodamage can be reversed by utilizing the ADSCs/their secretory factors alone [5, 7, 39], or in combination with other devices minimizing unwanted effects [42]. Identification of active proteins will be the next goal, and drug development using these proteins will suggest better strategies for skin aging in the future.
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32. Chen L, et al. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS ONE. 2008;3:e1886. 33. Baregamian N, et al. IGF-1 protects intestinal epithelial cells from oxidative stressinduced apoptosis. Surg Res. 2006;136:31–37. 34. Rahman ZA, et al. Antioxidant effects of glutathione and IGF in a hyperglycaemic cell culture model of fibroblasts: some actions of advanced glycaemic end products (AGE) and nicotine. Endocr Metab Immune Disord Drug Targets. 2006;6:279–286. 35. Shibuki H, et al. Expression and neuroprotective effect of hepatocyte growth factor in retinal ischemia-reperfusion injury. Invest Ophthalmol Visual Sci. 2002;43:528–536. 36. Tsao YP, et al. Pigment epithelium derived factor inhibits oxidative stress-induced cell death by activation of extracellular signalregulated kinases in cultured retinal pigment epithelial cells. Life Sci. 2006;79:545–550. 37. Kida H, et al. Protective effect of IL-6 on alveolar epithelial cell death induced by hydrogen peroxide. Am J Physiol. 2005;288:342–349. 38. Liochev SI, et al. How does superoxide dismutase protect against tumor necrosis factor: a hypothesis informed by effect of superoxide on ‘‘free’’ iron. Free Radic Biol Med. 1997;23:668–671. 39. Kang SH, et al. Improvement of melasma and scars with the secretory factors from ADSCs. Korean J Dermatol. 2007;45(Suppl. 2):136. 40. Kang WH, et al. Melasma: histopathological characteristics in 56 Korean patients. Br J Dermatol. 2002;146:228–237. 41. Kim EH, et al. The vascular charateristics of melasma. J Dermatol Sci. 2007;46:111–116. 42. Park BS, et al. Rejuvenation of aging skin using fractional CO2 laser resurfacing followed by topical application of ADSC protein extract. Kor J Dermatol. 2008;46(Suppl 1):266–267. 43. Matsumoto D, et al. Cell-assisted lipotransfer: supportive use of human adipose-derived cells for soft tissue augmentation with lipoinjection. Tissue Eng. 2006;12:3375–3382.
13 Aging and Intrinsic Aging: Pathogenesis and Manifestations Hanan Assaf . Mohamed A. Adly . Mahmoud R. Hussein
Introduction Cutaneous aging is a complex biological phenomenon consisting of two components: intrinsic aging and extrinsic aging. Intrinsic aging is also termed true aging which is an inevitable change attributable to the passage of time alone and is manifested primarily by physiologic alterations with subtle but undoubtedly important consequences for both healthy and diseased skin and is largely genetically determined [1]. Extrinsic aging is caused by environmental exposure, primarily to UV light, and more commonly termed photoaging. In sun-exposed areas, photoaging involves changes in cellular biosynthetic activity that lead to gross disorganisation of the dermal matrix [2]. The intrinsic rate of skin aging in any individual can be dramatically influenced by personal and environmental factors, particularly the amount of exposure to ultraviolet light. Photodamage, which considerably accelerates the visible aging of skin, also greatly increases the risk of cutaneous neoplasms. So, the processes of intrinsic and extrinsic aging are superimposed. As the population ages, dermatological focus must shift from ameliorating the cosmetic consequences of skin aging to decreasing the genuine morbidity associated with problems of the aging skin. Therefore, a better understanding of both the intrinsic and extrinsic influences on the aging of the skin, as well as distinguishing the retractable aspects of cutaneous aging (primarily hormonal and lifestyle influences) from the irretractable cutaneous aging (primarily intrinsic aging), is very important to solve the problem of aging [2].
Pathogenesis of Intrinsic Aging Logic dictates that one or more molecular events must underlie the aging process. These changes are now beginning to be unraveled and are discussed. As these mechanisms are identified, further insights into the underlying processes of skin aging should emerge and better strategies to prevent the undesirable effects of age on skin appearance should follow. The process of intrinsic
skin aging resembles that seen in most internal organs and an explanation is thought to involve decreased proliferative capacity leading to cellular senescence, and altered biosynthetic activity of skin derived cells [2]. The molecular mechanisms partly underlying skin aging comprise a multifaceted process influenced by various factors affecting different body sites at variable degrees. A stochastic process that implies random cell damage as a result of mutations during metabolic processes due to the production of free radicals is also implicated [2–4]. As the molecular mechanisms leading to human senescence are complex processes, different research approaches are used to study aging including studies of monogenic segmental progeroid syndromes. Two progeria syndromes, Werner’s syndrome (WS) and Hutchinson-Gilford progeria syndrome (HGPS), which are characterized by clinical features mimicking physiological aging at an early age, provide insights into the mechanisms of natural aging. They suggest a model of human aging. Based on recent findings on WS and HGPS, human aging can be triggered by two main mechanisms: telomere shortening and DNA damage. In telomere-dependent aging, telomere shortening and dysfunction may lead to DNA damage responses which induce cellular senescence. In DNA damage-initiated aging, DNA damage accumulates, along with DNA repair deficiencies, resulting in genomic instability and accelerated cellular senescence. In addition, aging due to both mechanisms (DNA damage and telomere shortening) is strongly dependent on p53 status. These two mechanisms can also act cooperatively to increase the overall level of genomic instability, triggering the onset of human aging phenotypes [3, 5]. Data from another trial revealing the molecular changes of intrinsic skin aging were analyzed by applying ‘‘Serial Analysis of Gene Expression’’ (SAGE(TM)) to skin biopsies of young and aged donors. The analysis resulted in several hundred differentially expressed genes with varying statistical significance. Of these, several genes were identified that either have never been described in skin aging before (e.g., APP) or have no identified function (e.g., EST sequences). This is the first time that
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_13, # Springer-Verlag Berlin Heidelberg 2010
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intrinsic skin aging has been analyzed in such a comprehensive manner, offering a new and partially unexpected set of target genes that have to be analyzed in more detail in terms of their contribution to the skin aging process [4, 6]. Moreover, normal human fibroblasts undergoing serial passaging have been extensively used to identify genes linked with aging. Most of the isolated genes relate to growth retardation signals and the failure of homeostasis that accompanies aging and senescence. In contrast, there is still limited knowledge regarding the nature of the genes that influence positively the rate of aging and longevity. Healthy centenarians represent the best example of successful aging and longevity. Studies using samples from these individuals have proved very valuable for identifying a variety of factors that contribute to successful aging [5, 7].
Manifestation of Skin Aging Various expressions of intrinsic aging include smooth, thinning skin with exaggerated expression lines. Extrinsically aged skin is characterized by photodamage as wrinkles, pigmented lesions, patchy hypopigmentations, and actinic keratoses [2].
The Wrinkle and Its Measurement There is a new method for the measurement of the size and function of the wrinkle, called ‘‘Profilometric’’ method. Facial wrinkles are not a single groove, but comprise an anatomical and functional unit (the ‘‘Wrinkle Unit’’) along with the surrounding skin. This Wrinkle Unit participates in the functions of a central neuromuscular system of the face responsible for protection, expression, and communication. Thus, the Wrinkle Unit, the superficial musculoaponeurotic system (superficial fascia of the face), the underlying muscles controlled by the CNS and Psyche, are considered to be a ‘‘Functional Psycho-Neuro-Muscular System of the Face for Protection, Expression and Communication’’. The three major functions of this system exerted in the central part of the face and around the eyes are: (1) to open and close the orifices (eyes, nose, and mouth), contributing to their functions; (2) to protect the eyes from sun, foreign bodies, etc.; and (3) to contribute to facial expression, reflecting emotions (real, pretended, or theatrical) during social communication. These functions are exercised immediately and easily, without any opposition (‘‘Wrinkling Ability’’) because of the presence of the Wrinkle Unit that gives (a) the site of refolding (the wrinkle is a waiting fold, ready to respond quickly at any moment for any skin
mobility need) and (b) the appropriate skin tissue for extension or compression (this reservoir of tissue is measured by the parameter of WTRV). The ‘‘Wrinkling Ability’’ of a skin area is linked to the wrinkle’s functions and can be measured by the parameter of ‘‘Skin Tissue Volume Compressed around the Wrinkle’’ in cubic millimetre per 30 mm wrinkle during maximum wrinkling. The presence of wrinkles is a sign that the skin’s ‘‘Recovery Ability’’ has declined progressively with age. The skin’s ‘‘Recovery Ability’’ is linked to undesirable cosmetic effects of aging and wrinkling. This new Profilometric method can be applied in studies where the effectiveness of anti-wrinkle preparations or the cosmetic results of surgery modalities are tested, as well as in studies focused on the functional physiology of the Wrinkle Unit [6]. Nevertheless, the gradual physiologic decline of aging skin is well documented [7, 8].
Aging of the Epidermis The most striking and consistent histologic change is flattening of the dermoepidermal junction, a considerably smaller surface between the two compartments and presumably less communication and nutrient transfer [9]. Dermal–epidermal separation has been demonstrated to occur more readily in old skin under experimental conditions. Inter-rete epidermal thickness probably remains constant with advancing age, but variability in epidermal thickness and in individual keratinocyte size increases [9]. Average thickness and degree of compaction of the stratum corneum appears constant with increasing age, although individual corneocytes become larger. The skin surface pattern examination reveals slight age-associated loss of regularity. There is also an age-related decrease in the barrier function of intact stratum corneum as measured by percutaneous absorption of at least some substances. Subsequent work suggests that age effects on percutaneous absorption depend, in part, on drug structure, with hydrophilic substances being less well absorbed through the skin of old subjects, but hydrophobic substances being equally well absorbed [1]. More recent work indicates that there is an age-associated decrease in percutaneous absorption for hydrophilic substances like hydrocortisone and benzoic acid, but no change for hydrophobic substances like testosterone and estradiol [10]. An age-associated decrease in epidermal turnover rate of approximately 30–50% between the third and eighth decades has been determined by a study of desquamation rates for corneocytes at selected body sites. The thymidine-labeling index of the epidermis in vivo has been reported to decline nearly 50% with age [1].
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There is also a corresponding 100% prolongation in stratum corneum replacement rate and a decrease in the linear growth rates for hair and nails. A study of epidermal wound healing showed that the repair rate in the skin linkwise declines with age, as restoration of normal skin surface markings in deroofed subcorneal blister sites required a median of approximately 3 weeks in subjects aged 18–25 years, but 5 weeks in subjects 65–75 years. Healing was essentially complete in all young subjects who were healed by 7 weeks, and the last by 8 weeks [11]. Clinical observations suggest that the development of chronic wounds frequently associate with persistent low tissue oxygen supply (hypoxia). The prolonged tissue hypoxia exposes wounds to bacterial infection, a prolonged inflammatory response, and eventually tissue necrosis. The elderly population accounts for a large portion of this morbidity [12]. Consistent with clinical observations, compelling evidence from laboratory studies has shown that age affects wound healing in several aspects: sprouting of aged microvessels was significantly less than the sprouting of young microvessels [13], increased gelatinase and collaginase levels in skin of aged donors and in wound fluid from chronic leg ulcers, and decreased TIMP (tissue inhibitor of matrix metalloproteinase) levels in the skin of aged donors as well as reduced deposition of matrix components and re-epithelialization [14–16]. A novel study about wound healing with increasing age done by Yu-Ping Xia et al. (2001) [17] revealed that keratinocytes isolated from elderly donors, in contrast to those from young individuals, had depressed migratory activity when they were exposed to hypoxia [17]. Analysis of underlying biochemical changes demonstrated a differential activation of matrix metalloproteinases by hypoxia in keratinocytes isolated from young and old ages. Matrix metalloproteinases-1 and Matrix metalloproteinases-9 and tissue inhibitor of matrix metaloproteinases-1 were strongly upregulated by hypoxia in young cells, whereas no induction was observed in aged cells. Furthermore, transforming growth factor-b1 signaling appears to be involved in keratinocyte differential response to hypoxia, as transforming growth factor-b type 1 receptor was upregulated by hypoxia in young cells, while there was no induction in aged cells. Transforming growth factor-b neutralizing reagents blocked hypoxia-induced matrix metalloproteinase-1, matrix metalloproteinase-9 expression, and hypoxia-induced cell migration as well. These results introduced by Yu-Ping Xia et al. (2001) [17] suggest that an age-related decrease in response to hypoxia plays a crucial part in the pathogenesis of related re-epithelialization in wounds [17]. A decrease in the number of the enzymatically active melanocytes per unit surface area of the skin,
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approximately 10 20% of the remaining cell population each decade, has been repeatedly documented [8]. It is not known whether the cells truly disappear or simply become undetectable by ceasing to produce pigment, but in either case the protective barrier of the body against ultraviolet radiation presumably is reduced. The number of melanocytic nevi (moles) also progressively decreases with age from a peak of 15 40 in the third and fourth decade to an average of four per person after age 50; moles are rarely observed in persons above age 80 [18]. The contribution of extracellular matrix components to intrinsic skin aging has been investigated thoroughly; however, there is little information as to the role of the cytoskeletal proteins in this process. Therefore, the new studies highlight the importance of the cellular compartment in this process and demonstrate that special attention has to be given to RNA as well as protein normalization in aging studies. OeNder et al. (2008) demonstrated that the mRNA levels of the genes for K1, K3, K4, K9, K13, K15, K18, K19 and K20 are downregulated in aged skin, K5 and K14 are unchanged, and K2, K16 and K17 are upregulated in aged skin. The mRNA data were confirmed on the protein level. This diverse picture is in contrast to other cytoskeletal proteins including components of the desmosome (JUP), microtubuli (TUBA) and microfilaments (ACTB) – often regarded as house-keeping genes – that were all reduced in aged skin [19]. The incidence of cancers, infectious diseases, and autoimmune disorders increases with advancing age [20]. In addition, aging is accompanied by a number of changes in immune function such as decreased lymphocyte proliferative responses to both mitogens and antigens, reduced delayed type hypersensitivity reactions, and decreased antibody responses to vaccination and infection [19]. Murine models of aging have demonstrated that there is an age-associated dysregulation in cytokine production, as evidenced by consistently decreased production of interleukin-2 (IL-2) and generally increased production of interleukin-4 [21]. These data, coupled with the increased incidence of cancer and the recurrence of latent viral infection in aged humans, have led to the hypothesis that the process of aging per se induces a switch from a predominantly type 1 cytokine (IL-2, INF-g, IL-12) profile supporting a dominant cellmediated immune response, to a predominantly type 2 cytokine (IL-4, IL-5, IL-6, IL-10) profile, promoting a dominant humoral response. A 20 50% reduction in the number of morphologically identifiable epidermal Langerhans cells occurs between early and late adulthood and may account in part for the age-associated decrease in immune responsiveness observed in the skin [7, 8].
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In principle, inhibition of aging should delay cancer. But the question which arises is whether it is possible to slow aging. As recently proposed, the nutrient-sensing TOR (target of rapamycin) pathway is involved in cellular and organismal aging. In rodents, certain conditions that interfere with the TOR pathway slow aging and prevent cancer. Retrospective analysis of clinical data reveals that in animals, from worms to mammals, caloric restrictions, life-extending agents, and numerous mutations that increase longevity all converge on the TOR pathway. And, in humans, cell hypertrophy, hyper-function and hyperplasia, typically associated with activation of TOR, contribute to diseases of aging. Theoretical and clinical considerations suggest that rapamycin may be effective against atherosclerosis, hypertension and hyper-coagulation (thus, preventing myocardial infarction and stroke), osteoporosis, cancer, autoimmune diseases and arthritis, obesity, diabetes, macula-degeneration, Alzheimer’s and Parkinson’s diseases. Finally, the extended life span will reveal new causes for aging (e.g., ROS, ‘‘wear and tear’’, Hayflick limit, stem cell exhaustion) that play a limited role now, in relation to TOR. So, there is a potential clinical use of TOR inhibitors in order to slow aging and delay cancer [22]. Richardson et al. (2004) also emphasized that regulation of growth and proliferation in higher eukaryotic cells results from an integration of nutritional, energy, and mitogenic signals [23]. Biochemical processes underlying cell growth and proliferation are governed by the phosphatidylinositol 3-kinase (PI3K) and TOR signaling pathways. The importance of the interplay between these two pathways is underscored by the discovery that the TOR inhibitor rapamycin is effective against tumors caused by misregulation of the PI3K pathway. Moreover, one of the recent breakthrough studies in TOR signaling resulted in the identification of the tuberous sclerosis complex gene products, TSC1 and TSC2, as negative regulators for TOR signaling. Furthermore, the discovery that the small GTPase Rheb is a direct downstream target of TSC1-TSC2 and a positive regulator of the TOR function has significantly advanced the understanding of the molecular mechanism of TOR activation. So, the regulation of TOR signaling is very important to control cell growth during normal development and tumorigenesis [24]. Vitamin D production, which is an important endocrine function of human epidermis, is suspected to decline with age. With advancing age, bone mass decreases markedly, especially in postmenopausal women, predisposing to trabicular bone fractures. Osteoporosis, or lack of cortical and trabicular bone, is a prominent factor, but some elderly individuals also have osteomalacia, since the decreased mineralization of bone is classically associated
with vitamin D deficiency. Although avoidance of dairy products, the principal dietary source of vitamin D, insufficient sun exposure, and sun screen use, undoubtedly contribute to vitamin D deficiency in the elderly [25]. The level of epidermal 7-dehydrocholesterol per unit skin surface area also appears to decrease linearly by approximately 75% between early and late adulthood suggesting that lack of its immediate biosynthetic precursor may also limit vitamin D production. In one study, old adult volunteers exposed to total body UV irradiation produced far less vitamin D3, over the ensuing week, than did complexion-matched young adult volunteers exposed to the same UV dose [2]. Neoplasia is associated with aging in virtually all organ systems, but is especially characteristic for aged skin. Acrochorrdon, cherry angioma, seborrheic keratosis, lentigo, sebaceous hyperplasia, one or more of these benign epidermal tumors is present in nearly every adult beyond age 65 years, and most individuals have dozens of lesions [20]. Actinically induced basal cell carcinoma and squamous cell carcinoma are by far the most common human malignancies. These benign and malignant neoplasms almost certainly reflect in part the loss of proliferative homeostasis with age.
Epidermal Proteins and Skin Aging Recent studies revealed by gel electrophoresis of healthy human skin (sun protected skin) from donors of different ages that there are age-associated changes in five proteins, which were identified as Keratin 10, Involucrin, Prealbumin, Hsp 27, and Rho B. More recently, it was found by the tool of immunohistochemistry that these epidermal proteins are expressed in the human skin epidermis and that their expression patterns undergo age-associated changes [26]. Examination of cryosections from healthy sun-protected skin derived from individuals ranging from the first decade of life until the ninth decade revealed that Keratin 10, Involucrin, Prealbumin, Hsp 27, and Rho B proteins are present in high density nearly in all layers of young epidermis (> Fig. 13.1). In contrast, the expression of the investigated epidermal proteins is reduced as a function of age. In some skin specimens from very old individuals, the proteins were not detectable [26]. All five proteins are known to be closely related to differentiation and proliferation of keratinocytes. Moreover, prealbumin, Hsp27 and Rho B have been demonstrated to play a crucial role in tumor cell biology. Thus, decreased expression of these proteins could be
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. Figure 13.1 Expression of Hsp27 protein in normal human skin. A moderate to strong expression is found in most epidermal layers, including the stratum corneum. a: Tyramid signal amplification (TSA) technique (200x), b: Avidin-Biotin complex (ABC) technique (200x). (Reprinted with permission from Adly et al. [47].) ß 2006, Elsevier
one reason for the increased prevalence of skin cancer in old individuals. Additionally, these proteins are possible marker proteins for intrinsic aging of the epidermis [26]. Additionally, Hsp27 and other epidermal proteins were found to be expressed in the human skin hair follicles and involved in hair follicle cycle control [26]. (> Fig. 13.2).
Aging of the Dermis Loss of dermal thickness approaches 20% in elderly individuals, although in sun-protected sites significant thinning occurs only after the eighth decade [27]; the remaining tissue is relatively acellular and avascular [1, 9]. Precise histologic concomitants of wrinkling, if any, are unknown, although the age-related loss of normal elastin fibers may be contributory [9]. Deep expression lines seem to result from contractions of connective tissue septae within the subcutaneous fat. In one study, an approximately 50% reduction in mast cells and a 30% reduction in venular cross-sections was noted in the papillary dermis of buttock skin from elderly adults compared to that from young adult controls, associated with a corresponding reduction in histamine released and other manifestations of the inflammatory response following UV radiation exposure [7]. The striking age-associated loss of vascular bed, especially of the vertical capillary loops that occupy the dermal papillae in young
skin, is felt to underlie many of the physiologic alterations in old skin. Reduction in the vascular network surrounding hair bulbs and eccrine, apocrine, and sebaceous glands may contribute to their gradual atrophy and fibrosis with age [1]. An age-associated decrease in dermal clearance of trans-epidermally absorbed materials has been reported and is probably due to alterations in both the vascular and extracellular matrix. A previous study showed that wheal resorption after intradermal saline injection required almost twice as long on average in elderly versus young adult subjects [1]. Controversely, the time required for development of a tense blister after topical application of 50% ammonium hydroxide is nearly twice as long in older subjects, suggesting a decreased transduction rate with age in injured skin. Impaired transfer of cells as well as solutes between the extravascular and intravascular dermal compartments is suggested by several studies to occur by age, but it is difficult to isolate these components in a complex inflammatory reaction. Decreased vascular responsiveness in the skin of older individuals has been documented clinically assessing vasodilation and transudation after application of standardized irritants, histamine, and the mast cell degranulating agent 48/80 [11]. Intensity of erythema following a standardized UV exposure is also decreased with age in normal skin, although factors other than decreased vascular responsiveness may also contribute [7]. A previous study that assessed cutaneous vascular response to the vasodilator
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. Figure 13.2 Expression of Hsp27 protein in normal human scalp skin anagen VI hair follicle, immunostained with (ABC) and (TSA) techniques. a and d show the distal region (200x), b and e show the central region (200x), c and f show the proximal bulb region (200x). D: shows schematic representation of anagen VI hair follicle. (Reprinted with permission from Adly et al. [47].) ß 2006, Elsevier
methyl nicotinate concluded that there was no difference between young adult and old adult subjects after correction for rate of drug absorption [28]. Compromised thermoregulation, which predisposes the elderly to sometimes fatal heat stroke, or hypothermia may be due in part to reduced vascularity of dermal arterioles, and in the latter instance, to loss of subcutaneous fat as well. The skin of healthy older subjects is less sensitive to dinitrochlorobenzene (DNCB) and to standard recall antigens, compared to the skin of young adult controls [29]. This decrease undoubtedly reflects the well documented decrease in total number of circulating thymus derived lymphocytes and in their responsiveness to standard mitogens. The elastic fibers in the skin are less well studied, but have been reported to show progressive cross linkage and
calcification with age in adult skin. On average, older skin has thicker elastic fibers than young skin, and elastic fiber alterations extend deeper into the dermis with advancing age [30, 31]. Small cysts and lacuna are common in aging elastic fibers, sometimes progressing to complete fragmentation. Similar changes can be produced experimentally by incubation of dermal slices with elastase or chymotrypsin (but not collagenase) in vitro, suggesting that enzymatic degradation of elastin may be a mechanism of normal dermal aging [30, 31]. The dermal microvasculature in middle-aged or elderly subjects may show mild vascular wall thickening; vascular wall thinning to less than half of the normal young adult measurement, associated with absent or reduced perivascular veil cells that has been reported in skin of very old subjects and probably contributes to vascular fragility [30, 31].
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Significant decrease in thrombomodulin-positive cells and vascularity were evidenced in the aged group. Specific subsets of the dermal dendrocyte populations and the blood microvasculature appear affected by aging. Capsaicin may limit these aging effects. [32]. Biochemical changes in collagen, elastin and dermal ground substance that have been described during foetal and early postnatal development are far greater than those that have been described with advancing age during adulthood. With advancing adult age, rat tail collagen does manifest a slight increase in the force of contraction (isometric tension), when heated above its shrinkage temperature, consistent with increasing cross-linkage of the collagen molecule [33]. Both rat tail tendon and human skin display a progressive decrease in the ratio of soluble to insoluble collagen [34]. The predominant cross-links in skin have been reported to decrease and virtually disappear with age in mature animals, however, using techniques that measure borohydride reducible cross-links, despite evidence of increasing mechanical stability. This suggests that some collagen cross-links in vivo may be progressively reduced or oxidized and are, therefore, no longer measurable. Certain non enzymatic cross-links in connective tissue, such as histidinoalanine and the Millard reaction product, do show a strong positive correlation with adult age, and have been suggested to contribute to age-associated changes in the dermis [1]. The proportion of recently synthesized dermal collagen, as determined by neutral salt extraction, is small and does not vary with age in adult [35, 36]. However, there is a significant decrease with age in the percent of total collagen that is released by pepsin digestion, and hence incompletely cross-linked, from approximately 25% at age 30 years to approximately 10% at age 75 years with a proportionate increase in the percent of insoluble collagen from approximately 70 88%. The amount of ketoaminelinked glycosylation of insoluble dermal collagen also increases with age, possibly related to slower collagen turnover or higher average glucose levels in the tissue [35, 36]. Prolyl- and lysyl-hydroxylase, enzymes necessary for intercellular stabilization of the collagen triple helix and for its intermolecular cross-linking, show an ageassociated decline in activity in human skin, although these coenzyme activities in cultured dermal fibroblasts from donors ranging in age from a few months to 94 years do not. This apparent contradiction could be explained by an age-associated decrease in either dermal fibroblast number in vivo or fibroblast responsiveness to a serumderived enzyme stimulating factor in vitro. There is some data concerning the possible postmaturational age-associated changes in mucopolysaccharides
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(glycosaminoglycans and proteoglycans) or other molecules of the ground substance in which collagen and elastic fibers are embedded. There appears to be a slight decrease with age in mucopolysaccharide content relative to dry weight or collagen content of the skin, especially for hyaluronic acid. Although mucopolysaccharides constitute only 0.1 0.3% of dry weight for whole skin, their decrease may adversely influence skin turgor, as proteoglycans bind a high volume of water in the dermis [37, 38]. Mechanical properties of the skin also change with age. Uniaxial and biaxial tension tests performed on excised abdominal skin stripes demonstrate progressive loss of elastic recovery, consistent with gradual destruction of the dermal elastin network, and the time required for excised skin to return to its original thickness after 50% compression is markedly prolonged [39]. This early work, which has been confirmed and extended by in vivo studies of ventral forearm skin of 133 volunteers in each decade of life, showed linear declines of approximately 25% in both men and women for elasticity and extensibility. Loss of elasticity began in childhood and continued through the ninth decade, while extensibility was constant through the sixth decade and then declines more rapidly thereafter. Overall, a picture emerges of aging dermis as an increasingly rigid, inelastic and unresponsive tissue, less capable of undergoing modification in response to stress. IGF-I is a key regulator of human skin aging and declining IGF-I levels with age may play a significant role in the reduction of skin surface lipids and thickness [40].
Nerves and Appendages By the end of the fifth decade, approximately half the population has at least 50% grey (white) body hair with an even higher proportion of depigmented scalp hair, and virtually everyone has some degree of greying due to progressive and eventually total loss of melanocytes from the hair bulb. Loss of melanocytes is believed to occur more rapidly in hair than in skin because the cells proliferate and manufacture melanin at maximal rates during the anagen phase of the hair cycle, while epidermal melanocytes are comparatively inactive throughout their lifespan. Scalp hair may gray more rapidly than other body hair because its anagen to telogen ratio is considerably greater than that of other body hair. Advancing age is also accompanied by a modest decrease in number of hair follicles. Remaining hairs may be smaller in diameter and grow more slowly. The process called balding results primarily from the androgen-dependent conversion of the relatively dark thick scalp hairs to lightly pigmented short
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fine hairs similar to those on the ventral forearm. Bitemporal hair line recession begins during late adolescence in most women and virtually all men. Assessment of baldness is hampered by lack of a precise definition, but by certain criteria advanced bitemporal and occipital hair loss in men increases in prevalence, respectively from 20% and 3% at the end of the third decade to more than 60% by the seventh decade. Eccrine glands decrease approximately 15% in average number during childhood in most body sites. Spontaneous sweating in response to dry heat is further reduced by more than 70% in healthy old subjects as compared to young controls, attributable primarily to a decreased output per gland. Maximal sweat production has not been quantified in the elderly, but is almost certainly reduced and probably predisposes to heat stroke in this age group [41]. Similar studies have not been performed for apocrine glands, although the apparently decreased requirement for under-arm deodorants in the elderly suggests decreased function. Lipofuscin gradually accumulates with age in the secretory cells of both eccrine and apocrine glands. Sebaceous gland size and number appear not to change with age [9]. The exponential decrease in sebum production of approximately 23% per decade beginning in the second decade in both men and women, approximately 60% over the adult lifespan, is attributed to the concomitant decrease in production of gonadal or adrenal androgen to which sebaceous glands are exquisitely sensitive. The clinical effects of decreased sebum production, if any, are unknown. There is no direct relationship to xerosis or seborrheic dermatitis [42]. Pacinian and Meissner’s corpuscles, the cutaneous end organs responsible for pressure perception, light and touch, progressively decrease to approximately one third of their initial average density between the second and ninth decades of life and display greater size variation. There are very few histologically demonstrable aging related changes in Merkel corpuscles or in free nerve endings. Decreased sensory perception was documented in old skin more than 3 decades ago by several techniques. Cutaneous pain threshold has been reported to increase up to 20% with advancing age [43]. The available data do not permit differentiation among an age-associated increase in the prevalence of peripheral neuropathy, a true aging change in healthy subjects, increased rate of heat dispersion in old skin due to age-associated dermal alterations, an increased peripheral nerve threshold to painful stimuli, and an increased central threshold to pain perception [43]. The many psychological and social factors influencing an individual reaction to pain may also be presumed to vary with age. In any case, either decreased awareness of, or reaction to, noxious stimuli
would facilitate wounding and irritation of old skin. Sympathetic nervous system activity is altered in aging.
Aging and Skin Diseases Disorders of the skin are known to be common and bothersome in the elderly, but existing incidence and morbidity figures are suspect [44]. Few dermatologic disorders occur predominantly in the elderly, and none is restricted to this age group. Perhaps the prototypic disease of old skin is bullous pemphigoid, characterized by subepidermal blister formation with fixation of complement and immunoglobulins along the basement membrane. Its predilection for the elderly may partially explained by the age-associated increase in circulating autoantibodies and ease of dermal epidermal separation, although other autoimmune and blistering dermatoses are not more common in old age. Possibly age-associated changes in the basement membrane itself render it specifically vulnerable to the disease process. More than two thirds of herpes zoster cases occur after the fifth decade, with an age adjusted annual incidence rate of approximately 0.25% at 20 50 years vs. more than 1% at age 80 years [45]. Post-herpetic neuralgia, uncommon in patients less than 40 years old, occurs frequently in older patients; more than half of those beyond age 60 years. In one large series this altered response to varicella virus has been established, however no mechanism explaining this altered response to varicella virus has been found. Recurrent herpes simplex infection also involves reactivation of latent virus in regional ganglia and T cell-mediated host defenses, but is more common in young adults and indeed rare among immunocompetent elders. The general phenomenon of impaired wound healing in the elderly may account for slower resolution of the acute eruption, but its relevance, if any, to post-herpetic neuralgia is unclear [45]. Age-associated muting of the inflammatory response might indeed be expected to reduce the risk of neuralgia, since prophylactic use of anti-inflammatory corticosteriods is often successful. Xerosis, the dry rough quality of old skin, may be attributable to a subtle disorder of epidermal maturation, although histologic studies reveal little alteration of either the viable epidermis or the stratum corneum with age. Available data fails to support water loss [1], decreased stratum corneum lipids [42] or altered amino acid composition as etiologic factors [42]. The surface irregularity may also be attributed simply to slower transit of corneocytes through the stratum corneum, allowing accumulation of damage in situ. Similarly, there is no explanation
Aging and Intrinsic Aging: Pathogenesis and Manifestations
for the pruritus that often accompanies xerosis. Unsupported hypotheses include frequent penetration of irritants through an abnormal stratum corneum and an altered sensory threshold due to subtle neuropathy. Many dermatoses more commonly observed in the elderly reflect the higher prevalence of systemic diseases such as diabetes, vascular insufficiency, and various neurologic syndromes in this population. In the case of chronic leg ulcers, for example, healing of previously recalcitrant lesions can sometimes be achieved by use of neonatal epidermal allograft, postulated to elaborate needed growth factors and/or matrix materials that the surrounding senescent host epithelium is incapable of producing. The allegedly increased incidence of other disorders such as tinea pedis or seborrheic dermatitis may reflect reduced local skin care with subsequent exacerbation of previously unapparent problems, rather than an age-associated change in the skin itself. Alternatively, subtle changes in the immune status may be responsible, in analogy to the increased prevalence and severity of those disorders in patients with acquired immunodeficiency syndrome [1]. Reduced tolerance to systematically administrated drugs is well documented in the elderly due to the decrements in lean body mass and metabolism and renal excretion of the active ingredients [46]. Comparable data for topically applied medication do not exist, but it is tempting to postulate that retarded dermal clearance of absorbed material reduced dermal mass and cellularity, and possibly altered metabolic capacity may render old skin more susceptible to both beneficial and adverse effects of topical medications, or at least alter the optimal dosage frequency [1]. In the case of corticosteriod preparations, relative vascular unresponsiveness may render blanching of erythema as an unreliable indicator of other effects in old skin.
Conclusion A better understanding of both the intrinsic and extrinsic influences on the aging of the skin, as well as distinguishing the retractable aspects of cutaneous aging (primarily hormonal and lifestyle influences) from the irretractable cutaneous aging (primarily intrinsic aging), is very important to solve the problem of aging.
Cross-references > Degenerative
Changes in Aging Skin
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References 1. Balin AK, Pratt LA. Physiological consequences of human skin aging. Cutis. 1989;43(5):431–436. 2. Puizina-Ivic N. Skin aging. Acta Dermatovenerol Alp Panonica Adriat. 2008;17(2):47–54. 3. Ding SL, Shen CY. Model of human aging: recent findings on Werner’s and Hutchinson-Gilford progeria syndromes. Clin Interv Aging. 2008;3(3):431–444. 4. Holtkotter O, Schlotmann K, Hofheinz H, Olbrisch RR, Petersohn D. Unveiling the molecular basis of intrinsic skin aging(1). Int J Cosmet Sci. 2005;27(5):263–269. 5. Chondrogianni N, de CMSD, Franceschi C, Gonos ES. Cloning of differentially expressed genes in skin fibroblasts from centenarians. Biogerontology. 2004;5(6):401–409. 6. Hatzis J. The wrinkle and its measurement – a skin surface Profilometric method. Micron. 2004;35(3):201–219. 7. Gilchrest BA, Stoff JS, Soter NA. Chronologic aging alters the response to ultraviolet-induced inflammation in human skin. J Invest Dermatol. 1982;79(1):11–15. 8. Gilchrest BA. Age-associated changes in the skin. J Am Geriatr Soc. 1982;30(2):139–143. 9. Montagna W, Carlisle K. Structural changes in aging human skin. J Invest Dermatol. 1979;73(1):47–53. 10. Roskos KV, Maibach HI, Guy RH. The effect of aging on percutaneous absorption in man. J Pharmacokinet Biopharm. 1989;17(6): 617–630. 11. Grove GL. Age-related differences in healing of superficial skin wounds in humans. Arch Dermatol Res. 1982;272(3–4):381–385. 12. Van de Kerkhof PC, Van Bergen B, Spruijt K, Kuiper JP. Agerelated changes in wound healing. Clin Exp Dermatol. 1994;19(5): 369–374. 13. Arthur WT, Vernon RB, Sage EH, Reed MJ. Growth factors reverse the impaired sprouting of microvessels from aged mice. Microvasc Res. 1998;55(3):260–270. 14. Ashcroft GS, Horan MA, Ferguson MW. Aging is associated with reduced deposition of specific extracellular matrix components, an upregulation of angiogenesis, and an altered inflammatory response in a murine incisional wound healing model. J Invest Dermatol. 1997;108(4):430–437. 15. Ashcroft GS, Herrick SE, Tarnuzzer RW, Horan MA, Schultz GS, Ferguson MW. Human ageing impairs injury-induced in vivo expression of tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -2 proteins and mRNA. J Pathol. 1997;183(2):169–176. 16. Ashcroft GS, Kielty CM, Horan MA, Ferguson MW. Age-related changes in the temporal and spatial distributions of fibrillin and elastin mRNAs and proteins in acute cutaneous wounds of healthy humans. J Pathol. 1997;183(1):80–89. 17. Xia YP, Zhao Y, Tyrone JW, Chen A, Mustoe TA. Differential activation of migration by hypoxia in keratinocytes isolated from donors of increasing age: implication for chronic wounds in the elderly. J Invest Dermatol. 2001;116(1):50–56. 18. Maize JC, Foster G. Age-related changes in melanocytic naevi. Clin Exp Dermatol. 1979;4(1):49–58. 19. Oender K, Trost A, Lanschuetzer C, Laimer M, Emberger M, Breitenbach M, Richter K, Hintner H, Bauer JW. Cytokeratin-related loss of cellular integrity is not a major driving force of human intrinsic skin aging. Mech Ageing Dev. 2008;129(10):563–571. 20. Blagosklonny MV. Prevention of cancer by inhibiting aging. Cancer Biol Ther. 2008;7(10):1520–1524.
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21. Albright JW, Mease RC, Lambert C, Albright JF. Trypanosoma musculi: tracking parasites and circulating lymphoid cells in host mice. Exp Parasitol. 1999;91(2):185–195. 22. Blagosklonny MV. Aging: ROS or TOR. Cell Cycle. 2008;7(21): 3344–3354. 23. Richardson CJ, Schalm SS, Blenis J. PI3-kinase and TOR: PIKTORing cell growth. Semin Cell Dev Biol. 2004;15(2):147–159. 24. Inoki K, Ouyang H, Li Y, Guan KL. Signaling by target of rapamycin proteins in cell growth control. Microbiol Mol Biol Rev. 2005; 69(1):79–100. 25. Matsuoka LY, Wortsman J, Hanifan N, Holick MF. Chronic sunscreen use decreases circulating concentrations of 25-hydroxyvitamin D. A preliminary study. Arch Dermatol. 1988;124(12): 1802–1804. 26. Hussein MR. Analysis of p53, BCL-2 and epidermal growth factor receptor protein expression in the partial and complete hydatidiform moles. Exp Mol Pathol. 2009. 27. de Rigal J, Escoffier C, Querleux B, Faivre B, Agache P, Leveque JL. Assessment of aging of the human skin by in vivo ultrasonic imaging. J Invest Dermatol. 1989;93(5):621–625. 28. Roskos KV, Bircher AJ, Maibach HI, Guy RH. Pharmacodynamic measurements of methyl nicotinate percutaneous absorption: the effect of aging on microcirculation. Br J Dermatol. 1990;122(2): 165–171. 29. Wayne SJ, Rhyne RL, Garry PJ, Goodwin JS. Cell-mediated immunity as a predictor of morbidity and mortality in subjects over 60. J Gerontol. 1990;45(2):M45–48. 30. Braverman IM, Fonferko E. Studies in cutaneous aging: II. The microvasculature. J Invest Dermatol. 1982;78(5):444–448. 31. Braverman IM, Fonferko E. Studies in cutaneous aging: I. The elastic fiber network. J Invest Dermatol. 1982;78(5):434–443. 32. Quatresooz P, Pierard GE. Immunohistochemical clues at aging of the skin microvascular unit. J Cutan Pathol. 2009;36(1):39–43. 33. Escoffier C, de Rigal J, Rochefort A, Vasselet R, Leveque JL, Agache PG. Age-related mechanical properties of human skin: an in vivo study. J Invest Dermatol. 1989;93(3):353–357.
34. Miyahara T, Murai A, Tanaka T, Shiozawa S, Kameyama M. Agerelated differences in human skin collagen: solubility in solvent, susceptibility to pepsin digestion, and the spectrum of the solubilized polymeric collagen molecules. J Gerontol. 1982;37(6):651–655. 35. Schnider SL, Kohn RR. Effects of age and diabetes mellitus on the solubility and nonenzymatic glucosylation of human skin collagen. J Clin Invest. 1981;67(6):1630–1635. 36. Schnider SL, Kohn RR. Effects of age and diabetes mellitus on the solubility of collagen from human skin, tracheal cartilage and dura mater. Exp Gerontol. 1982;17(3):185–194. 37. Lipson MJ, Silbert JE. Acid mucopolysaccharides of tadpole tail fin and back skin. Biochim Biophys Acta. 1965;101(3):279–284. 38. Kondo K, Seno N, Anno K. Mucopolysaccharides from chicken skin of three age groups. Biochim Biophys Acta. 1971;244(3):513–522. 39. Daly CH, Odland GF. Age-related changes in the mechanical properties of human skin. J Invest Dermatol. 1979;73(1):84–87. 40. Makrantonaki E, Vogel K, Fimmel S, Oeff M, Seltmann H, Zouboulis CC. Interplay of IGF-I and 17beta-estradiol at age-specific levels in human sebocytes and fibroblasts in vitro. Exp Gerontol. 2008;43 (10):939–946. 41. Silver AF, Chase HB. An in vivo method for studying the hair cycle. Nature. 1966;210(5040):1051. 42. Downing DT, Stewart ME, Strauss JS. Changes in sebum secretion and the sebaceous gland. Dermatol Clin. 1986;4(3):419–423. 43. Procacci P, Zoppi M, Maresca M. Experimental pain in man. Pain. 1979;6(2):123–140. 44. Beauregard S, Gilchrest BA. A survey of skin problems and skin care regimens in the elderly. Arch Dermatol. 1987;123(12):1638–1643. 45. Hope-Simpson RE. The Nature of Herpes Zoster: A Long-Term Study and a New Hypothesis. Proc R Soc Med. 1965;58:9–20. 46. Vestal RE. Aging and pharmacology. Cancer. 1997;80(7):1302–1310. 47. Adly MA, Assaf HA, Hussein MR. Expression of the heat shock protein-27 in the adult human scalp skin and hair follicle: hair cycle-dependent changes. J Am Acad Dermatol. 2006;54(5):811–817.
38 Aging and Melanocytes Stimulating Cytokine Expressed by Keratinocyte and Fibroblast Mutsumi Okazaki
Introduction
Fibroblast
In the skin pigmentation, the actinic damage plays a major role [1], but the effect of chronologic cellular aging is also an important factor. The chief cellular components of the skin other than melanocytes are keratinocytes and fibroblasts, whose paracrine effects on melanocytes (rather than melanocyte itself) play an important role in the epidermal pigmentation [2–11]. Human keratinocytes express several melanogenic cytokines, such as endothelin-1 (ET-1) [2–4], granulocyte macrophage colony stimulating factor (GM-CSF) [5], stem cell factor (SCF), and basic fibroblast growth factor (bFGF) [7–9]. Human fibroblasts, on the other hand, secrete several melanogenic cytokines such as bFGF, HGF, and SCF [6, 10, 11]. Further, interleukin-1a (IL-1a), a pro-inflammatory cytokine, stimulates the production of ET-1 by keratinocytes and of HGF by fibroblasts [3, 12–14]. It has been reported that the overexpression of these melanogenic cytokines is responsible for the age-related pigment ary cutaneous disorders [15–17]. The age-associated change was studied in cytokine secretion by keratinocytes and fibroblasts based upon this paracrine cytokine network within the skin for epidermal pigmentation mechanisms.
Normal skin specimens were taken from Japanese patients (disused skin during plastic surgery, i.e., after the dog-ear correction). Informed consent was obtained from all patients. Fibroblasts were cultured from 19 specimens (age = 26.7 15.6, from 7 to 65 year old, 8 males, 11 females). The methods of isolation and culture of fibroblasts were reported previously [18]. Fibroblasts were grown in the fibroblast growth medium (FGM), which consists of Dulbecco’s modified Eagle’s medium (DMEM), 0.6 mg/mL glutamine, and 10% fetal calf serum (FCS). Third cultures of fibroblasts were used for the experiments. Fibroblasts were seeded in a 60 mm culture dish at a density of 5 105 cells/5 mL and cultured in FGM. After human fibroblasts had been cultured for 96 h at 37 C under a 5% CO2 atmosphere, the medium was collected to quantify HGF, SCF, and bFGF, respectively, by ELISA.
Correlation Between Age and Secretion of Melanogenic Cytokine Studies were planned to elucidate whether the aging of keratinocytes and fibroblasts was related to the potential to secrete several melanogenic cytokines. In the first experiment, the keratinocytes and fibroblasts derived from the skin of different chronological ages were cultured, and the secretions of melanogenic cytokines were evaluated by ELISA (enzyme-linked immuno-sorbent assay). The series of study were carried out with the informed consent of the person whose skin samples were used.
Keratinocyte Normal skin specimens were obtained from Japanese patients, and keratinocytes were cultured from 16 specimens (age = 28.0 17.1, from 7 to 64 years, 6 males, 10 females). The methods of isolation and culture of keratinocytes were reported previously [18]. Keratinocytes were grown in the serum-free keratinocyte growth medium (KGM; Kyokuto Seiyaku, Tokyo) which consists of MCDB153 with high concentrations of amino acids, transferrin (final concentration 10 g/mL), insulin (5 g/mL), hydrocortisone (0.5 g/mL), phosphorylethanolamine (14.1 g/mL), and bovine pituitary extract (40 g/mL). The final concentration of Ca2+ in the medium was 0.03 mM. Second cultures of keratinocytes were used for the experiment. Keratinocytes were seeded in a 60 mm culture dish at a density of 1.5 105 cells/5 mL, and cultured in KGM supplemented with 0.5% FCS. After human
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_38, # Springer-Verlag Berlin Heidelberg 2010
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keratinocytes had been cultured at 37 C under a 5% CO2 atmosphere for 72 h, the keratinocyte-conditioned medium was collected to quantify IL-1a, ET-1, and GM-CSF, respectively, by ELISA. The comparison of the cytokine concentration between male and female was carried out using unpaired t-test. And the scatter diagrams showing the relationship between age and cytokine concentration were drawn, and simple linear regression equations were calculated, and simple linear regression test was used to determine whether there was any correlation between age and
concentration of cytokine. A value of P <0.05 was considered statistically significant. 1. Cytokine secretion by fibroblasts No gender differences in the donor age and cytokine concentration were found between male and female. There was no correlation between age and cytokine concentration (R: coefficient of determination; HGF: R = 0.0054, P = 0.98; SCF: R = 0.19, P = 0.44; bFGF: R = 0.0064, P = 0.98) (> Fig. 38.1a–c).
. Figure 38.1 Scatter diagram showing the relationship between donor age and value of cytokine concentration in fibroblasts. The lines represent the linear regression equation (n = 19; R, coefficient of determination). (a) HGF y = 159 0.034x, R = 0.0054, P = 0.98; (b) SCF y = 191 0.84x, R = 0.19, P = 0.44; (c) bFGF y = 2.62 0.00079x, R = 0.0064, P = 0.98
Aging and Melanocytes Stimulating Cytokine Expressed by Keratinocyte and Fibroblast
2. Cytokine secretion of keratinocytes No gender differences in the donor age and cytokine concentration were found between male and female. There was a significant correlation between age and IL-1a concentration (R = 0.71, P = 0.002). There was a relatively weak correlation between age and ET-1 concentration, but the correlation was not significant (R = 0.41, P = 0.051). No correlation existed between age and GM-CSF concentration (R = 0.32, P = 0.23) (> Fig. 38.2a–c).
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In the second experiment, the secretions of IL-1a and ET-1 by keratinocytes were compared between the second and fifth cultures. In this study, the fifth cultures of keratinocytes derived from nine donors who were less than 30 years old were used. Cell cultures and ELISA assay were performed in the same way as the first experiments. A comparison of the cytokine concentrations between the second and fifth culture keratinocytes was carried out using paired t-test. A value of P <0.05 was considered statistically significant.
. Figure 38.2 Scatter diagram showing the relationship between donor age and value of cytokine concentration in keratinocytes. The lines represent the linear regression equation (n = 16; R, coefficient of determination). (a) IL-1a y = 4.0 + 0.36x, R = 0.71, P = 0.002*; (b) ET-1 y = 11.0 + 0.24x, R = 0.41, P = 0.051; (c) GM-CSF y = 15.3 + 0.17x, R = 0.32, P = 0.23
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. Figure 38.3 Comparison of IL-1a and ET-1 secretion by keratinocytes between second and fifth cultures. Each ELISA values (values are means SD derived from three wells of each specimens) of nine donors and average values (AV) are shown. *P <0.005 to second cultures. (a) IL-1a; (b) ET-1
The concentration of IL-1a was significantly increased in fifth cultures compared with second cultures (P <0.005), but the levels of ET-1 did not differ significantly (P = 0.52) (> Fig. 38.3a, b).
Conclusion The results suggest that increasing secretion (occurring as the cell ages) of IL-1a by keratinocytes may play a role in the age-associated skin change. Because IL-1a stimulated the ET-1 production by keratinocytes [3], and ET-1 induced an increase in tyrosinase activity and stimulated melanocyte proliferation [2–4], the increasing secretion of IL-1a by keratinocytes may be responsible for the accentuated cutaneous pigmentation associated with
aging. In the study, however, the correlation between age and ET-1 secretion by keratinocytes was not significant, although there was a weak correlation (R = 0.41, P = 0.051). One of the possible reasons for this is small sample size, and another is that the autocrine stimulation by IL-1a might not be as efficient on the aged keratinocytes under the specific culture condition. This is supported by the second experiment; the levels of ET-1 secretion did not differ significantly, although IL-1a secretion was significantly increased in old (the fifth) cultures compared with young (the second) cultures. In the culture methods used, keratinocytes derived from aged skin (approximately older than 30 years of age) can no longer proliferate vigorously after three to four serial subcultures. In a preliminary study, the keratinocytes derived from 1-year-old child could be cultivated
Aging and Melanocytes Stimulating Cytokine Expressed by Keratinocyte and Fibroblast
serially for about seven to eight passages under these culture conditions when they are subcultured every 7 days. At seventh or eighth cultures, most of the cultures were composed of enlarged and flattened cells, and such cultures were considered senescent, and subculturing was not continued. These findings were consistent with those reported by Boyce and Ham [19], and the chronologic cellular age of the fifth culture can be estimated 30–40 years older than that of second culture. It was reported that after irradiation with ultraviolet B (UVB), ET-1 production by keratinocytes increased accompanied by significant secretion of IL-1a [3]. The study suggests that melanocytes in the aged skin are stimulated persistently by keratinocytes via IL-1a-ET-1 cascade, as if the skin were irradiated with UVB. Thus, epidermal pigmentation might be persistently accentuated in the aged skin. The study also suggests that there was no correlation between secretions of melanogenic cytokine by fibroblasts and cellular age. It was reported that human dermal fibroblasts from aged donors (over 80 years) produced more HGF than fibroblasts from young and middle-aged donors, and the production of HGF by human embryonic lung fibroblasts increased sharply after about 70% completion of their lifespan in culture, which is mainly due to autocrine stimulation by IL-1a [20]. IL-1a was reported to stimulate the expression of HGF in the fibroblasts
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[12–14]. In this study, however, there was no correlation between age and HGF secretion, probably because the age of donor ranged from 7 to 65 years old. One hypothesis concerning age-associated cutaneous pigmentation can be proposed (> Fig. 38.4); the secretion of IL-1a by keratinocytes gradually increase with age, which stimulates ET-1 production by keratinocytes autocrinely and HGF production by dermal fibroblasts paracrinely, and these cytokines stimulate melanocyte proliferation and induce an increase of tyrosinase activity in melanocytes. On the other hand, the production of IL-1a by fibroblast would increase abruptly from the age of about 70 to 80 years, which stimulates HGF production by dermal fibroblasts autocrinely and ET-1 production by keratinocytes paracrinely. One of the limitations of the study is that skin specimens were obtained from various sites of the body; sitespecific differences of cytokine secretion could not be assessed because of small number of specimen involved. It is an essential variable whether the specimen was obtained from sun-exposed or sun-protected sites. To exclude the influence of acute sunlight exposure to the skin on the cytokine secretion, the isolated skin cells were used in culture (not use freshly isolated cells). Conversely, it was inevitable that biological age of the cultured cells used in the study is larger than age of donors (as was
. Figure 38.4 Schematic showing the hypothesis of the accentuated pigmentation in the aged skin. The secretion of IL-1a by keratinocytes gradually increase with age, which stimulates ET-1 production by keratinocytes autocrinely and HGF production by dermal fibroblasts paracrinely, and these cytokines stimu late melanocyte proliferation and induce an increase of tyrosinase activity in mel anocytes. On the other hand, the production of IL-1a by fibroblast would increase abruptly from the age about 70–80, which stimulates HGF production by dermal fibroblasts autocrinely and ET-1 production by keratinocytes paracrinely
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mentioned above, it is estimated that 7 days’ culture of keratinocyte corresponds to about 10–12 years of age). Another limitation of the study is that only Japanese specimens were used. There are some reports describing the differences of pigmentation, or response to ultraviolet radiation in the skin of different ethnic groups [21, 22]. It is probable that the general tendency for age-related change in the melanogenic cytokine secretion is common in all ethic groups, but there may be some ethnic difference. Because cytokine expression was evaluated with ELISA (protein level) in the study, the expression of membrane-bound SCF (m-SCF) of keratinocyte was not assessed; in epidermal keratinocyte, SCF is expressed as a membrane-bound form, not in a secretory or soluble cytokine form such as ET-1 [23]. Even in stimulated conditions such as following UVB exposure, only the expression of m-SCF is accentuated in epidermal keratinocytes [23]. Evaluation of the expression of m-SCF, mRNA might reveal a different mechanism of accentuated cutaneous pigmentation. It was reported that accentuated epidermal pigmentation in lentigo senilis and seborrheic keratosis are attributed to a larger amount of secretion of ET-1 by keratinocytes [15, 17]. The study suggests the probability that formations of such lesions in the older person are associated with an increasing production of IL-1a by epidermal keratinocyte. Furthermore, because IL-1a is a primary mediator that responds to inflammation and injury [24], the transcription of genes involved in skin inflammation may be persistently induced in the aged skin. Thus, it is probable that the aged skin are always exposed to the conditions like inflammation, and the increased potential to secrete IL-1a in aged keratinocytes is associated with various skin aging in addition to the epidermal pigmentation.
Acknowledgment I wish to thank Kotaro Yoshimura, Gentaro Uchida, and Kiyonori Harii for their contribution as co-researcher, and Hiroshi Ohtsu and Junji Kishimoto for their statistical suggestions.
References 1. Leyden JJ. Clinical features of aging skin. Br J Dermatol. 1990; 122(35):1–3.
2. Yada Y, Higuchi K, Imokawa G. Effect of endothelins on signal transduction and proliferation in human melanocytes. J Biol Chem. 1991;266:18352–18357. 3. Imokawa G, Yada Y, Miyagishi M. Endothelins secreted from human keratinocytes are intrinsic mitogens for human melanocytes. J Biol Chem. 1992;267:24675–24680. 4. Imokawa G, Miyagishi M, Yada Y. Endothelin-1 as a new melanogen: coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis. J Invest Dermatol. 1995; 105:32–37. 5. Imokawa G, Yada Y, Kimura N, et al. Granulocyte/macrophage colony-stimulating factor is an intrinsic keratinocyte-derived growth factor for human melanocytes in UVA-induced melanosis. Biochem J. 1996;313:625–631. 6. Grabbe J, Welker P, Dippel E, et al. Stem cell factor, a novel cutaneous growth factor for mast cells and melanocytes. Arch Dermatol Res. 1994;161:78–84. 7. Halaban R, Langdon R, Birchall N, et al. Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes. J Cell Biol. 1988;107:1611–1619. 8. Puri N, van der Weel MB, de Wit FS, et al. Basic fibroblast growth factor promotes melanin synthesis by melanocytes. Arch Dermatol Res. 1996;288:633–635. 9. Scott G, Stoler M, Sarkar S, et al. Localization of basic fibroblast growth factor mRNA in melanocytic lesion by in situ hybridization. J Invest Dermatol. 1991;96:318–322. 10. Matsumoto K, Tajima H, Nakamura T. Hepatocyte growth factor is a potent stimulator of human melanocyte DNA synthesis and growth. Biochem Biophys Res Commun. 1991;176:45–51. 11. Imokawa G, Yada Y, Morisaki N, et al. Characterization of keratinocyte- and fibroblast-derived mitogens for human melanocytes – their roles in stimulated cutaneous pigmentation. Melanogenesis and malignant melanoma. In: Hori Y, Hearing VJ, Nakayama J (eds) Biochemistry, Cell Biology, Molecular Biology, Pathophisiology, Diagnosis and Treatment. Amsterdam: Elsevier Science B.V., 1996, pp. 35–48. 12. Matsumoto K, Okazaki H, Nakamura T. Up-regulation of Hepatocyte growth factor gene expression by interleukin-1 in human skin fibroblasts. Biochem Biophys Res Commun. 1992;188:235–243. 13. Maas-Szabowski N, Fusenig N. Interleukin-1-induced growth factor expression in postmitotic and resting fibroblasts. J Invest Dermatol. 1996;107:849–855. 14. Mildner M, Mlitz V, Gruber F, et al. Hepatocyte growth factor establishes autocrine and paracrine feedback loops for the protection of skin cells after UV irradiation. J Invest Dermatol. 2007;127: 2637–2644. 15. Kadono S, Manaka I, Kawashima M, et al. The role of the epidermal endothelin cascade in the hyperpigmentation mechanism of lentigo senilis. J Invest Dermatol. 2001;116:571–577. 16. Hattori H, Kawashima M, Ichikawa Y, et al. The epidermal stem cell factor is over-expressed in lentigo senilis: Implication for the mechanism of hyperpigmentation. J Invest Dermatol. 2004;122: 1256–1265. 17. Teraki E, Tajima S, Manaka I, et al. Role of endothelin-1 in hyperpigmentation in seborrheic keratosis. Br J Dermatol. 1996;135: 918–923. 18. Okazaki M, Yoshimura K, Uchida G, et al. Correlation between age and the secretions of melanocyte-stimulating cytokines in cultured keratinocytes and fibroblasts. Br J Dermatol. 2005; 153(s3):23–29.
Aging and Melanocytes Stimulating Cytokine Expressed by Keratinocyte and Fibroblast 19. Boyce ST, Ham RG. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J Invest Dermatol. 1983;81:33s–40s. 20. Miyazaki M, Gohda E, Kaji K, et al. Increased Hepatocyte growth factor production by aging human fibroblasts mainly due to autocrine stimulation by interleukin-1. Biochem Biophys Res Commun. 1998;246:255–260. 21. Tadokoro T, Yamaguchi Y, Batzer J, et al. Mechanisms of skin tanning in different racial/ethnic groups in response to ultraviolet radiation. J Invest Dermatol. 2005;124:1326–1332.
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22. Wagner JK, Parra EJ, L Norton H, et al. Skin responses to ultraviolet radiation: effects of constitutive pigmentation, sex, and ancestry. Pigment Cell Res. 2002;15:385–390. 23. Hachiya A, Kobayashi A, Ohuchi A, et al. The paracrine role of stem cell factor/c-kit signaling in the activation of human melanocytes in ultraviolet B-induced pigmentation. J Invest Dermatol. 2001; 116:578–586. 24. Murphy JE, Robert C, Kupper TS. Interleukin-1 and Cutaneous inflammation: a crucial link between innate and acquired immunity. J Invest Dermatol. 2000;114:602–608.
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26 Aging Genital Skin and Hormone Replacement Therapy Benefits William J. Ledger
Introduction
Vaginal Changes
The most precise word that can be used to describe menopause is change. The most dramatic alteration in body function for older women is the cessation of menses, the usual monthly flow of bloody vaginal fluid. This obviously easily perceived and recognized phenomenon announces a new era for aging women; a new life passage with a loss of fertility and a growing awareness with new symptoms of the inexorable process of aging. This chapter will focus upon the impact of this part of the woman’s life cycle upon the vagina and the vulva.
This diminishment in estrogen levels dramatically changes the vagina. There is a major decrease in the amount of vaginal secretion and a loss in both the elasticity and capacity of the vagina. These changes become very apparent to women. The annual or biannual visit to the gynecologist becomes an unwelcome chore, for it is increasingly uncomfortable. Socially, intercourse can change from a pleasurable sensation to a painful event with a subsequent drop in libido. Parallel to these unpleasant alterations, the vaginal pH changes from the normal acid range of 4.5 or less to an alkaline reading greater than 4.5. Upon microscopic examination of the vaginal secretion in a saline preparation, menopausal women have smaller, more rounded immature vaginal squamous cells and a markedly diminished number of acid-producing bacteria, such as the lactobacilli. (> Fig. 26.1) These microscopic changes in the bacterial flora can be confirmed by culture. One report shows a marked diminishment in the number of probiotic lactobacilli, with lactobacilli the dominant vaginal species in only 13% of the menopausal subjects [2]. Anaerobes become the dominant species. The numbers of these organisms were previously held in check by the dominance of acid-producing bacteria like the lactobacilli. There are clinical consequences of these changes in the vaginal bacterial flora that can result in lifestyle alterations for these women. Perimenopausal and particularly post-menopausal women are colonized by Escherichiae coli at an increased incidence, and this is inversely related to the presence of lactobacilli [3]. These vaginal bacterial changes make the woman much more susceptible to lower urinary tract infection due to E. coli [4]. The protective effect of the lactobacilli, keeping the numbers of these gram-negative aerobes in check, has been lost, and the relatively short female urethra becomes an easier transit site for these pathogens. The perimenopausal woman is also much more prone to suffer from a grossly purulent and persistent vaginitis, named Desquamative Inflammatory Vaginitis (DIV) [5]. The hallmarks of this troublesome syndrome,
Estrogen The lack of the female hormone estrogen brings down hormonal activity that helps maintain the health of lower genital tract tissue. This loss results in the many changes that confront post-menopausal women. This absence is not an abrupt shift, the curtain does not suddenly fall one evening as the human drama of reproductive life ends, with the new playbill, postmenopausal existence beginning the next day. Instead, perimenopause, the transition period’s new name, describes the very gradual changes in the years or decades before the cessation of menses. This is a slow and inexorable process in which minute bodily alterations occur over months and years that are gradually perceived by the woman involved. Although early on, menstruation continues unabated, there is a drastic falloff in reproductive success. Assisted reproductive physicians note a stunning drop after the age of 42 in the ability of women to become pregnant, and those few who succeed have a high number of spontaneous first-trimester pregnancy losses [1]. Subsequently, these perimenopausal women notice changes in their menstrual cycle, which can vary from too frequent and heavier periods, to less frequent and lighter periods. Along with these menstrual changes, these women become aware of ‘‘hot flashes,’’ i.e., vasomotor instability, sleeping trouble, a dry and less lubricated vagina that makes intercourse much less pleasurable.
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. Figure 26.1 Microscopic examination of vaginal secretions of an asymptomatic post-menopausal woman
. Figure 26.2 Superficial tissue changes of post-menopausal woman not on hormone replacement therapy
seen most commonly in women over the age of 40, are an alkaline pH, a negative whiff test when a drop of vaginal secretion is placed in a 10% potassium hydroxide mixture, the absence of lactobacilli, an increase in other bacterial forms, many immature vaginal squamous cells, and most significantly, an outpouring of inflammatory white cells. These vaginal changes have made older women more susceptible to urinary tract and vaginal infections.
Some women of this age group who are susceptible will develop a chronic inflammatory skin disease, lichen sclerosis, which predominantly affects the skin and mucous membranes of the vulvar and rectal area. This thinned vulvar skin also loses much of its natural surface defense mechanisms and is more likely to become and stay inflamed. This is an expected response. Post-menopausal women, not taking estrogen, have higher induced levels of the pro-inflammatory cytokine interleukin-1, interleukin-6, and tumor necrosis factor a when compared to women in their reproductive years [7]. The body’s response to this new inflammation is a repair process with new tissue formation. This new tissue regeneration in an inflammatory site is almost always accompanied by vulvar and perineal itching. This initiates a vicious cycle. The itching leads to scratching, often when the patient is asleep and unaware of her response. The scratching increases the tissue inflammation, and the cycle begins anew. This becomes a constant source of symptomatology leading to patient frustration. The thinning of the keratin layer of the labia majora and labia minora also diminishes the protection normally afforded against bacterial and fungal adhesion to this tissue site. The epithelial cells of reproductive-age women also produce many substances, such as peptides [8], that can kill potential bacterial or fungal pathogens. Lessened production of these substances in menopausal women increases the chances of infection. The resulting skin infections add to the inflammation. Vulvar fungal infections occur commonly in this aging population (> Fig. 26.3), and if not eradicated by treatment contribute to the cycle of inflammation,
Vulvar Changes There are visually apparent alterations in the vulva of postmenopausal women. The tremendous reduction in the levels of estrogen results in a loss of tissue elasticity and an obvious thinning of the vulvar tissue (> Fig. 26.2). This thinning, apparent to the naked eye, is accompanied by other significant subcutaneous changes. These become more noticeable when the V-600 Syris imaging system is used, which allows the observer to view the tissue two cell layers beneath the surface [6]. The thinning of the skin is highlighted. In addition, using a system of cross-polarized light visualization, there is decreased vascularity, more dryness, and more subdermal inflammation. On the surface, the vulvar skin becomes retracted with the tissue demarcations between the labia majora and labia minora becoming blunted. These changes are particularly apparent using the magnification of the colposcope. This thinner tissue area is much more fragile and more prone to splitting and cracking, at times leaving the distressed patient with a new symptom: a painful, slow-healing vulvar cut.
Aging Genital Skin and Hormone Replacement Therapy Benefits
. Figure 26.3 Candida Albicans infection of vulva of post-menopausal woman
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this response by using commercially available, over-thecounter lubricants can have unintended results. These products contain propylene glycol, a chemical preservative to which some women with inflamed mucous membranes can develop sensitivities. This contact dermatitis increases even more the local inflammation and pain. To avoid this possibility, these couples should be advised to use mineral oil or olive oil as a pre-coital lubricant. They are unquestionably messy, but do not dry out over time with firm residual kernels of commercial products that can be an additional source of irritation, and they contain no chemicals that could cause a local tissue reaction.
Outside Influences: Hormone Replacement Therapy (HRT)
itching, scratching, and more inflammation. Bacterial infection of the vulva can be a life-threatening problem in post-menopausal women, particularly in diabetics in whom synergistic bacterial vulvar infections can occur, with tissue death requiring operative intervention for survival [9].
Outside Influences: Male The television advertisements of the American pharmaceutical industry are dominated by concerns about both male urinary function and sexual prowess. The theme of male erectile dysfunction vies with difficulty in urinary voiding as a lead-in to capture the attention of the male drug consumer. The models in the ads blend the knowing smiles of the older, but still physically rugged and vigorous male with the ‘‘come hither’’ looks of their very attractive female partners. For the viewer, the obvious result will be the nirvana of continued sexual satisfaction. For the post-menopausal sexual partner of these newly invigorated older males, there can be a tremendous downside for vaginal and vulvar skin health. The resulting increase in heterosexual activity can cause vaginal and/or vulvar lacerations because of the dryness and decreased elasticity of the lower genital tract skin and mucous membranes. This genital tract pain sets in place a cascade of continuing problems. Pain will reduce vaginal secretion and the memory of prior discomfort can result in heightened pelvic floor muscle contraction when insertion is attempted, making a prior unpleasant sexual experience even more uncomfortable. Reflex attempts to modify
Since most of the changes in the health of the vagina and vulva in perimenopausal and menopausal women are related to a lessened production of estrogen, it would be logical to assume that estrogen supplementation would be an aid for these women. This is true, within limits. Estrogen therapy is most helpful as a preventive measure. It is usually beneficial if given before the aging vaginal and vulvar tissue changes of thinning, loss of turgor and elasticity have become grossly apparent. It either prevents or markedly slows these genital skin and mucous membrane alterations. In the vagina of women receiving estrogen supplementation, the pH is as acidic as it was in the reproductive years, and the dominant bacterial flora remains the acid-producing lactobacilli. If the progressive tissue changes of thinning, inflammation, or lacerations occur, the effects of estrogen are much less dramatic. They may accelerate the healing of these cuts and decrease inflammation, but they do not return the tissue to a premenopausal state. These are not formulae for the ‘‘fountain of youth,’’ but they do halt further deterioration. Other pharmaceutical agents, such as topical adrenocortical steroids, are much better in combating local tissue inflammation and are frequently prescribed as an alternative therapy in women with inflammatory vulvar changes. Despite these shortcomings, the lifestyle of women taking supplemental estrogen is usually much improved. They experience less discomfort with intercourse and have fewer lower urinary tract infections. All of these are positive observations. The reader should reflexively wonder why there has not been a deluge of television advertisements for the improvement in female well-being and pleasure with estrogen replacement therapy to parallel the onslaught of ads trumpeting male well-being. This would give equal time to the current focus upon male
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sexual satisfaction. Instead, there is an absence of these ads and market evidence of a decrease in the current use of hormone replacement therapy (HRT) in American women. Why? This phenomenon of the current less frequent use of HRT in the United States has been largely driven by the publicity surrounding the results of Women’s Health Initiative (WHI) studies. These studies, sponsored by the National Institutes of Health (NIH), have highlighted the potential dangers for women taking systemic HRT. There is a small but increased risk of developing breast cancer in women taking HRT for 5 years or more [10]. This had been previously reported in earlier studies [11]. Other unexpected adverse outcomes were noted in asymptomatic menopausal women taking HRT. They had a greater chance of having either a heart attack or a stroke as compared to the population of women taking a placebo [10]. This was a surprise, for prior studies of HRT had shown beneficial effects, with a lowering of blood lipids [12]. This had been the basis for the medical dogma of the 1980s that HRT had cardiovascular and cerebrovascular benefits for all post-menopausal women. These WHI studies raised doubts about that hypothesis and led to a precipitous drop in the numbers American women using HRT. There were some major problems in the WHI postmenopausal women study design that have been too often overlooked. The focus of the WHI study was upon asympotomatic menopausal women. This in itself was appropriate, for if symptomatic women had been studied, there would have been an inordinate loss of women recruited for the study who were given the placebo. Their symptoms would have continued, and they would have been more likely to drop out of the study. Using asymptomatic women avoided this, but to recruit this population, one third of the women had been menopausal for over 5 years and another third had been menopausal for over 10 years. This fact should raise some concerns about the interpretation of the study. This is not the usual target group for HRT. The focus should be upon women who become symptomatic with perimenopause, with symptoms becoming more pronounced as they become menopausal. For the skin, the greatest benefits from estrogen treatment result from the prevention or slowing of the skinaging process and not from the treatment of established aging skin changes. This goal of HRT therapy – prevention, not treatment – was not reached in two thirds of the WHI study population. An interesting aside is that the minority one-third of these WHI study patients who received HRT within the first 5 years of menopause did not have the
increased cardiovascular and cerebrovascular risks noted in the other two-thirds. There are dangers of the use of systemic HRT for women, particularly an increased risk of breast cancer [13], but these have been over-emphasized to include local estrogren products while downplaying the benefits. Many women with vaginal or vulvar symptoms due to skin aging will benefit from a wide variety of local estrogen products, including creams, vaginal tablets, or a vaginal ring, none of which to date have been associated with any of the systemic risks that have been attributed to systemic HRT. The concerns about HRTs have been too widely publicized. All too often, many women will avoid these potentially beneficial local medications for they have been led to believe that the risks associated with systemic HRT apply to all estrogen products. This is unfortunate, because for many women, these agents improve their sense of worth for they help to modify at least some of the pitfalls of aging. Most of the older women using estrogen look and feel better. They are a selected population, for they were symptomatic prior to the use of these hormones. For them, the hormones improve their daily lives and the quality of their social interactions. These benefits for women should be cited as they have been for men. Males, taking drugs for erectile dysfunction, have an increased risk of heart attacks and blindness, both serious medical problems. These have not been the primary focus in discussion as have been the concerns about the risks for HRT for women.
Conclusion There is a gender gap here. For women, downplay benefits and stress the risks; for men, emphasize pleasure and gloss over the risks. This is a curious commentary upon the current American scene.
Cross-references > Biological
Effects of Estrogen on Skin Aging and Oral Hormone Replacement
> Climacteric
Therapy
References 1. American Society for Reproductive Medicine. Age and Fertility: A Guide for Patients (Patient Information Series). Birmingham: American Society for Reproductive Medicine, 2003.
Aging Genital Skin and Hormone Replacement Therapy Benefits 2. Hillier SL, Lau RJ. Vaginal microflora in post-menopausal women who have not received estrogen replacement therapy. Clin Infect Dis. 1997;25(Suppl 2):S123–126. 3. Pabich WL, Fihn SD, Stamm WE, et al. Prevalence and determinants of vaginal flora alternations in post-menopausal women. J Infect Dis. 2003;188:1054–1058. 4. Raz R, Stamm WE. A controlled trial of intravaginal estriol in postmenopausal women with recurrent urinary tract infection. N Engl J Med. 1993;329:753–756. 5. Sobel JD. Desquamative inflammatory vaginitis: a new subgroup of purulent vaginitis responsive to topical 2% clindamycin therapy. Am J Obstet Gynecol. 1994;171:1215–1220. 6. Farage M, Singh M, Ledger WJ. Investigation of the sensitivity of a cross-polarized light visualization system to detect subclinical erythema and dryness in women with vulvovagintis. Am J Obstet Gynecol. 2009;201:1e1–1e6. 7. Pfeilschifter J, Kodtiz R, Pfohl M, et al. Changes in proinflammatory cytokine activity after menopause. Endocr Rev. 2002;23:90–119.
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8. Hancock REW. Lationic peptides: effectors in innate immunity and novel antimocrobials. Lancet Infect Dis. 2001;1:156–164. 9. Addison WA, Livengood CH III, Hill GB, et al. Necrotizing fasciitis of vulvar origin in diabetes patients. Obstet Gynecol. 1984;63: 473–479. 10. Rossouw TE, Anderson GL, Prentice RL, et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomize controlled trial. JAMA. 2002;288:321–333. 11. Hulley S, Grady B, Bush T, et al. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in post-menopausal women. JAMA. 1998;280:605–613. 12. Rossouw JE, Prentice RL, Manson JE, et al. Postmenopausal hormone therapy and risk of cardiovascular disease by age and years since menopause. JAMA. 2007;297:1465–1477. 13. Chlebowski RT, Kuller LH, Prentice RL, et al. Breast cancer after use of estrogen plus progestin in post-menopausal women. N Engl J Med. 2009;360:573–582.
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19 Aging of Epidermal Stem Cells Alexandra Charruyer . Ruby Ghadially
Introduction Advances in aging biology indicate that stem cells have a crucial role in organ maturation and aging. Studies have demonstrated molecular and biochemical changes in tissue-resident progenitor cells and their microenvironments during chronological aging of tissues such as the heart [1], brain [2], and hematopoietic system [3]. In this chapter knowledge in the field of aging and stem cells derived from tissues other than the epidermis is reviewed, and the challenges of studying aging stem cells discussed. Subsequently, epidermal stem cells are reviewed and changes in progenitor populations of the epidermis that occur with age discussed. Finally, the body of knowledge specifically related to the aging of epidermal stem cells and the implications of stem cell aging for carcinogenesis are examined.
Aging and Stem Cells Changes in Stem Cell Frequency with Aging Information about the impact of aging on stem cells has been obtained from the hematopoietic system and it remains an ideal system for this type of study as stem and progenitor cells are most well defined in the hematopoietic system. The loss of immune function and the increased incidence of myeloid leukemia associated with aging were thought to be due to a decrease in hematopoietic stem cell frequency. Research has now contradicted this assumption. Several studies indicate that murine hematopoietic stem cell numbers increase substantially with age [4, 5]. Limiting dilution assays have shown that hematopoietic stem cells from aged mice were more efficient at myeloid reconstitution than hematopoietic stem cells from young mice. Aged hematopoietic stem cells were found to be five times as numerous, but one quarter as efficient at engrafting, as young hematopoietic stem cells, suggesting a small increase in reconstitution ability [5]. In another study, the relative number of the most primitive stem cell was found to be three- to fourfold higher in aged over young mice but there was a decrease in the proliferative activity
of aged hematopoietic stem cells [4]. These studies show that there is an increase in primitive precursors and a decrease in proliferative ability with aging. In skeletal muscle, estimates of stem cell (satellite cell) number have produced varying results. One study looking at DNA content and nuclei count by electron microscopy demonstrated an increase in the satellite cell number in aging rats [6]. Using flow cytometry and CD34 expression there was no difference in satellite cell frequency in aged and young murine muscles [7]. Finally, a microscopic study of skeletal muscle reported a decrease in satellite cells during aging [8]. Thus, a consensus has yet to be reached regarding the effect of aging on skeletal stem cell frequency.
Intrinsic Cellular Modifications in Stem Cells with Aging While changes in stem cell frequency play a role in aging, there is also evidence for intrinsic cellular modifications in stem cells with aging. It is challenging to distinguish intrinsic cellular aging from the effects of the cellular milieu when stem cells are studied in their natural environment, and the isolation of a pure stem cell population is needed (for review see [9]). Epidermal stem cells isolated from young and old mice based on their Hoechst dye exclusion were analyzed for gene expression profile by cDNA arrays. There was similar gene expression of 422 genes assayed in young and old epidermal stem cells [10]. Expression profiles of highly purified long-term repopulating hematopoietic stem cells showed that aging was associated with a down-regulation of genes mediating lymphoid function and up-regulation of genes involved in myeloid fate, indicating that the loss of immune function and the increase in leukemia in the elderly is due to intrinsic alterations in hematopoietic stem cells [11]. These latter studies provide evidence that the intrinsic properties of stem cells change with age.
Changes in the Stem Cell Niche with Aging Stem cell homeostasis is maintained not only by intrinsic factors, but also by extrinsic factors, such as the local
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environment of the stem cell niche, the surrounding tissue, the systemic milieu of the organism and the external environment (for review see [9, 12]). Age-related modifications of extrinsic factors may include alterations in the composition of the extracellular matrix, in membrane proteins and lipids, and in factors that constitute the systemic milieu. Modifications in the stem cell niche during the aging process have been addressed in Drosophila. The Drosophila germline stem cells together with the somatic cells present in the niche is one of the most well-defined stem cell niches [13]. In somatic niche cells from older Drosophila testes there is a decrease in expression of a cell adhesion molecule (DE-cadherin) and a self-renewal ligand (unpaired). This was correlated with an overall decrease in stem cell numbers inside the niche [14]. Furthermore, restoring self-renewal (unpaired) resulted in an increase of the number of germline stem cells in older males compared with age-matched controls. Thus modifications of the somatic cells that constitute the niche in aged testes, can affect the frequency of stem cells inside the niche. Furthermore, murine spermatogonial stem cells could be serially transplanted in young mice recipients without showing any decline in stem cell number or colony forming ability for more than 3 years, indicating that a young environment can influence stem cell self renewal capacity, and that the failure of niche integrity plays a key role in the reproductive deficit in aged mice [15]. The effect of environment on skeletal muscle satellite cells has been studied [16]. No significant differences were found in mass or maximum force between old muscles grafted into young hosts and young muscle grafted into those same young hosts. Conversely, young muscles grafted into old recipients did not regenerate better than old muscles grafted into the same old hosts, indicating an important role for the environment in muscle regeneration after transplantation. Conditioned medium from differentiated myotubes of young mice exhibited a strong mitogenic action on aged satellite cells in vitro, whereas no mitogenic action was observed from conditioned media of myotubes from aged mice, either on young or on aged satellite cells [17] (for review see [18]). More recently, systemic influences on aged satellite cells were investigated using parabiotic pairings in which regenerating tissues in aged animals were exposed either to their own serum or that of young mice (isochronic or heterochronic parabioses respectively) [19]. In this study, exposing injured muscles from old mice to heterochronic parabioses greatly improved muscle regeneration and myotubule formation was similar to that observed in young mice. The authors concluded that a young systemic environment
could improve the impaired regenerative ability of aged skeletal stem cells. These studies demonstrate the important influence of both the local and systemic environment within which stem cells reside.
Symmetric and Asymmetric Stem Cell Division and Aging Stem cells in the niche undergo two types of division, symmetric self-renewal divisions leading to two identical daughter cells and asymmetric divisions leading to one daughter cell identical to the original stem cell and another non-stem daughter cell that leaves the niche and undergoes differentiation [13, 20]. It is predicted that aged stem cells would have preserved or increased self-renewal potential (symmetrical divisions) and decreased asymmetrical divisions, and in order to maintain a constant rate of cellular production the aged will have more proliferation in the transit amplifying cells [21, 22]. Studies agree with this prediction; in different stem cell compartments including the hematopoietic system and the intestinal crypt there is an increase in transit amplifying cells with age [23] (for review see [21]).
Challenges of Studying Aging and Stem Cells Issues surrounding the study of aging include the study of aging versus development, the study of animals of an appropriate age, the heterogeneity of aged animals, and the lack of a pure population of stem cells from most tissues. Many studies are confounded by the use of neonatal versus adult or neonatal versus aged tissue. Such studies may not reflect changes of aging, but rather changes occurring during development from birth to adulthood. It is important to keep these studies distinct, and the focus here is on aging of epidermal stem cells from the adult to aged individual. In aging studies, the age of the aged animals to be studied is of great importance, and guidelines for such studies are limited. The definition of aging varies depending on the physiological system under question. For example, many age-related changes in the immune system are evident by the 70% survival point and even earlier, while kidney changes start later. The most common age used to model old age is somewhere around the 50% survival point, although it may vary from the 70% survival point to the 30% survival point. It has been stated that ‘‘without epidemic disease or exaggerated or lopsided
Aging of Epidermal Stem Cells
tumor incidence, the 50% survival point can be considered as an indicator of the onset of the senescent period’’ [24]. It is important to note that results from extremely aged animals are not reliable because they may be the consequence of advanced disease rather than aging. The best approach, although often difficult in practice, is to study multiple time points during the senescent period [25]. Aging is also difficult to study because of the heterogeneity that is associated with the aging process. Indeed, the variance in vital characteristics in the elderly is substantially higher compared with other groups of the population [26]. One universal characteristic of aging is the accumulation of molecular damage, which induces alterations in gene expression, genomic instability, mutations, tissue disorganization and organ dysfunction. Because of the low probability that two molecules will be damaged in the same way and with the same intensity substantial molecular heterogeneity results, and this leads to clinical heterogeneity in the elderly population [27]. While hematopoietic stem cells can be isolated at a single cell level [28], epidermal stem cell markers that allow isolation of epidermal stem cells at a single cell level are yet to be found. Studying populations of stem cells that are of unknown purity poses a challenge. Furthermore, the lack of a good understanding of the stem cell hierarchy in epidermis complicates studies further. In summary, studies of aging and in particular aged epidermal stem cells are profoundly affected by the choice of age for the young cohort, the choice of age for the aged cohort, the heterogeneity of the aged cohort, and the lack of a pure epidermal stem cell population for study.
Characterization of Epidermal Stem Cells The integrity of the epidermis is maintained by division of cells in the proliferative basal layer that replace differentiated cells in the outermost stratum corneum layer. Not all proliferative cells in the basal layer are stem cells. Transit-amplifying cells amplify the basal cell population but are limited to a finite number of divisions before they differentiate and are lost from the epidermis [29]. Stem cells of the basal layer are responsible for maintaining and generating the adult epidermis and its appendages, including hair follicles and sebaceous glands. Different stem cell niches have been described: (1) the follicular stem cell that resides in the hair follicle bulge [30, 31] (for review see [32]), (2) the interfollicular stem cell [33] (for review see [34]), and (3) the melanocyte stem cell localized in the hair follicle [35].
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Phenotypic analysis of hematopoietic stem cells has provided the ability to separate the long-term proliferating cell from the less primitive cells detected in colonyforming assays [36]. These types of studies have allowed a hierarchy of hematopoietic stem cell differentiation to be determined. Different methods to isolate epidermal stem cells have been proposed, although very little has been achieved in defining a hierarchy of progenitors in the epidermis. Several of the most prevalent methods for isolating putative epidermal stem cells that have been used to study aging of epidermal stem cells are discussed here (> Table 19.1), including (1) side population (SP) cells that efflux Hoechst 33342 fluorescent dye [37, 38], (2) integrin a6 bright/CD71 dim human keratinocytes [39, 40], (3) collagen adhesion, and (4) a quantitative epidermal regeneration assay [33, 41]. Other methods/ markers such as label retaining cells [42], p63 [43], keratin 19 [44], keratin 15 [45] and elevated levels of b catenin [46] have also been reported as putative stem cell markers. Hoechst dye exclusion is an example of a method borrowed from other tissues that may be useful for isolating epidermal stem cells. Hematopoietic and muscle stem cells are identified by the ability of stem cells to exclude Hoechst 33342 dye [47, 48]. A multidrug resistance P-glycoprotein pump present in stem cells mediates this exclusion. This multidrug resistance pump is also associated with resistance to anti-cancer drugs and is over expressed in some cancer cell lines [49]. Basal epidermal cells also express this P-glycoprotein [50]. Cells showing Hoechst exclusion were defined as stem cells by their clonal ability, high proliferative potential, and the ability to recapitulate an epidermis in vitro [51]. Transit amplifying cells were identified as cells showing a medium forward and orthogonal scatter without regard to Hoechst exclusion. These cells could not maintain an epidermis in vitro [51]. A recent study of mouse epidermis, characterizing side population and non-side population cells showed that a6 integrin, b1 integrin, Sca-1, keratin 14, and keratin 19 were all highly expressed by side population cells, while CD34, CD71, and E-cadherin were more weakly expressed by side population cells than by non-side population cells. This demonstrates that side population cells express previously established stem cell associated proteins [52]. In contrast, it was found that side population keratinocytes are distinct from the label-retaining cell population, since side population cells and label retaining cells showed distinctly different and non-overlapping expression profiles of b1 and a6 integrins [37]. Thus although the frequency of epidermal stem cells in the Hoechst dye excluding population is unknown, this population
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. Table 19.1 Prevalent methods for isolating putative epidermal stem cells Method Side population
Description
References
Cells that efflux Hoechst 33342 dye are highly clonogenic and recapitulate an epidermis in vitro.
Terunuma et al. [37], Triel et al. [38]
Integrin Quiescent cells. Li A, Simmons PJ, a6brightCD71dim High long-term Kaur P. [39], proliferative Terunuma et al. [40] capacity. Produce human skin equivalents with a stratified epidermis. Collagen adhesion
Cells that adhere rapidly to type IV collagen are highly proliferative in vitro. Can form a robust stratified epidermis in vitro.
Watt FM. [55], Jones PH, Harper S, Watt FM. [56], Kaur et al. [57]
In vivo epidermal repopulating unit
In vivo Schneider et al. [33], transplantation Strachan et al. [41], model (in chambers, Charruyer et al. [78] or by subcutaneous injection). Individual keratinocytes regenerate a cornified, stratified epithelium (epidermal repopulating unit/ ERU) for the long term.
Others
Label retaining cells, p63, keratin 19, keratin 15, b catenin.
Bickenbach JR. [42], Yang et al. [43], Stasiak et al. [44], Lyle et al. [45], Zhu AJ, Watt FM. [46]
contains cells with phenotypic attributes of stem cells and high proliferative potential in vitro. High a6 integrin and low transferrin receptor (CD71) expression are perhaps the most accepted epidermal stem cell markers to date [39, 53]. The a6 integrin bright, CD71 dull cells are relatively quiescent and populations of these cells have very high long-term proliferative capacity [39]. Tani et al observed that a6briCD71dim murine dorsal keratinocytes were a quiescent population of small cells, with
a high nuclear to cytoplasmic ratio, consistent with primitive cells. In addition, 1.4% of total isolated keratinocytes were both a6briCD71dim and label retaining cells [53]. In human skin, the a6briCD71dim population contained smaller cells with a high nuclear to cytoplasmic ratio, was capable of producing a high number of large colonies after 10 days of culture, and produced skin equivalents with a stratified and thick epidermis [54]. In summary, although the stem cell purity is unknown, in both human and mouse epidermis the a6briCD71dim population contains undifferentiated cells with colony forming ability in vitro. Determining how in vitro assays relate to stem cell behavior in vivo is complex and much of the evidence is inconsistent with the concept that a colony forming cell is a stem cell [33, 41]. Nonetheless the analysis of colony formation in vitro is an attractive and prevalent procedure used to define stem cell behavior. b1 integrin is expressed in all basal keratinocytes. As keratinocytes leave the basal layer they down-regulate the expression of b1 integrin [55]. Cells that adhere rapidly to a b1 integrin ligand, type IV collagen, were found to have a high proliferative potential in vitro, whereas cells that adhere slowly divide only a few times before all of their progeny undergo terminal differentiation [56]. Rapidly adherent cells also form a robust stratified epidermis in vitro [54]. 20–40% of basal cells have high b1 integrin expression, which is in great excess of the proportion of basal cells that are estimated to be stem cells in vivo and thus it is likely that high b1 integrin expression does not uniquely select for epidermal stem cells [56]. It is believed that stem cells cannot be defined based solely on their proliferative behavior in culture [57]. Stem cell populations lack the appropriate stimuli for growth in vitro and surrogate in vitro analyses to assess putative stem cell behavior may not accurately distinguish between epidermal stem cells and progenitors cells [41]. A quantitative epidermal regeneration assay was developed that involved the use of dissociated keratinocytes allowed to regenerate a cornified, stratified epithelium on top of dermal fibroblasts seeded onto the subcutaneous fascia of immunodeficient mice. By seeding progressively lower numbers of GFP-positive keratinocytes in this repopulation assay, limiting dilution analysis quantifies the frequency of cells with long-term repopulating ability in a given population. Chambers are kept for 9 weeks [33]. The value of an in vivo assay for epidermal stem cells is well recognized [41, 58]. Using the quantitative in vivo regeneration assay described above it is possible to quantify epidermal stem cells from different keratinocyte populations. The study of epidermal stem cells from different sources such as aged versus young or diseased versus
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healthy would be greatly enhanced by the availability of pure populations of epidermal stem cells. To date techniques have been developed for enriching populations of keratinocytes for early progenitors, but not at the single cell level. Given the difficulty in finding specific stem cell markers, investigation is needed to determine combinations of markers that can enrich for epidermal stem cells at a single cell level. Markers found in stem cells from other tissues, embryonic stem cell markers, or even cancer stem cell markers found in tumorigenic tissues may provide useful strategies for the isolation of normal epidermal stem cells at a single cell level.
Aging and the Keratinocyte Proliferative Compartment Abundant historical studies in both human and animal models demonstrate that aging of the epidermis is accompanied by decreased proliferation, both basally and in response to proliferative stimuli [59–66]. These studies have led to the concept that epidermal stem cells, which are responsible for the maintenance of the epidermis, are involved in the aging process. The effect of aging on the keratinocyte proliferative compartment (stem cells and transit amplifying cells) has been studied in vivo. After skin injury, the regeneration of tissue requires stem cell mobilization [30, 67], which has made wound healing a valuable model for the study of the impact of aging on epidermal stem cells. A battlefield surgeon performed an early study of wound healing and age during World War I [68]. Using a cicatrisation index, the biological age of an injured soldier could be determined by measuring the rate of closure of war wounds. Soldiers in their thirties healed more slowly than those in their twenties. Standardized superficial skin wounds were created in young adults (18–25 years) and aged adults (65–75 years) and healing was monitored. By day 28, the younger group had completely restored their original skin markings, while the older cohort took double the time (56 days) [62]. A nonradioactive method, using Dansyl chloride that binds only to nonviable corneocytes, was used to measure stratum corneum transit time. Transit time was increased in aged persons and there was no difference in the number of horny layers, indicating that the increase in stratum corneum transit time in the aged was a reflection of decreased epidermal proliferation [63]. These studies of stem cells and transit amplifying cells provide some information on how the proliferative compartment of epidermis changes with aging in vivo, but not specifically on how the stem cell changes.
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The effect of aging on keratinocyte proliferation has also been studied using in vitro models of aging. Rheinwald and Green compared the proliferative behavior of seven human newborn-derived keratinocyte cultures with 3-, 12- and 34-year-derived cultures [69]. Newborn-derived cultures were able to undergo 25– 51 cell generations versus 20–27 for older person-derived cultures and could be maintained through three to six passages versus two to three passages. In addition, plating efficiency (colony forming ability) was up to 15.7% for the newborn versus 0.7% for the older person-derived cultures. Another study using newborn human keratinocytes compared with adult human keratinocytes reported that while attachment rate is independent of donor age, plating efficiency was strongly dependent on donor age. Plating efficiency was 2–10% in newborn cultures and below 0.01% in adult cultures [61, 70]. It should be noted that the above in vitro studies may reflect development, aging, or a combination of development and aging since they used newborn human keratinocytes.
Aging and Epidermal Stem Cells The above in vivo and in vitro observations of aging and proliferation were made on the entire keratinocyte population and not on different proliferative subpopulations. To address the differences in behavior of proliferative keratinocyte subpopulations (stem cells versus transit amplifying cells), the growth potential of individual proliferative clones from different donors (two neonatal, one 64-year-old, and one 78-year-old) was studied [71]. After plating individual cells, resultant clones were passaged into a second dish. The original clone was classified by the appearance of cells in the secondary dish into holoclones (cells that form large rapidly growing colonies), paraclones (that form uniformly small, terminal colonies) and meroclones (that form both types of colonies). With age, the number of holoclones decreased and the number of paraclones increased when compared with newborns. The authors concluded that the culture lifetime of a keratinocyte population declines with the age of the donor, as demonstrated by the change in the proportion of the three clone types. After studying the effects of aging on keratinocyte proliferation in vitro, and with the study of stem cell frequency in the hematopoietic system, and the discovery of epidermal stem cell markers, several studies have now addressed the influence of aging on epidermal stem cell number. Such studies have produced varying results. There were twice as many stem cells in neonatal mouse
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epidermis (1–2 days) as in adult (8–14 weeks) (8.4% versus 3.8%, respectively), using small size and Hoechst fluorescence to define a stem cell [72]. In a study of four human foreskins (from 1, 4, 35 and 61 year old donors), there was a decrease in the number of putative epidermal stem cells in adult versus neonatal human epidermis, as defined by a6 integrinhi CD71lo expression [73]. Such comparisons of adult and neonatal samples may reveal changes of development and/or aging. However, while neonatal murine epidermis had three times as many Hoechst dye-excluding cells as adult epidermis, the number of cells that could exclude Hoechst dye was unchanged in aged adult versus young adult murine epidermis [10]. The epidermal stem cells from young adults and aged adults had similar characteristics in culture, similar gene expression, and did not show extensive loss of telomeres with age. In the same study, epidermal stem cells isolated from 22-month-old transgenic mice that expressed GFP were injected into mouse blastocysts. Six months later various tissues of the resultant mice contained GFP positive cells, demonstrating that the developmental potency of murine epidermal stem cells is not altered with aging. It was concluded from this study that epidermal stem cells are resistant to cellular aging [10]. Furthermore, human and mouse keratinocytes that exclude Hoechst dye showed little variability in protein expression profiles in aged versus neonatal epidermis, suggesting that as epidermal stem cells age they do not substantially change their cellular characteristics [74]. The differing findings presented above could result from the study of keratinocytes of varying ages, their human or murine derivation, the possibility that different putative epidermal stem cells markers are not isolating the same population of progenitor cells, or that different methods have different efficacy in isolating epidermal stem cells. While the previous in vitro studies are very informative, skin aging has been difficult to study in vivo due to the lack of relevant models. A recent study attempted to address this issue by studying the effect of aging on keratin 15 positive progenitors from young (2–6 months) and aged (22–26 months) mice [75]. Using whole-mount immunostaining, they observed a similar number of keratin 15 positive bulge stem cells in follicles of both young and aged mice, suggesting that epidermal stem cell frequency is not affected by aging. Surprisingly, there was only a modest, and not statistically significant, decrease in proliferation as measured by Ki67. This is different from previous in vitro studies, and suggests that in vivo assays may produce different results in the study of skin aging and epidermal stem cells.
In order to examine in vivo, whether the decreased proliferative ability of aged epidermis could be explained by either quantitative and/or qualitative alterations in the stem and/or transit amplifying cell proliferative compartments, a quantitative in vivo transplantation assay was used similar to the hematopoietic assays that have been informative about changes in hematopoietic progenitors with aging [5, 76, 77]. In vivo transplantation assays of aged and young adult keratinocytes showed that while no significant difference in epidermal stem cell frequency could be detected, transit amplifying cell frequency was greater in the aged. With aging there was both an increased growth fraction (proportion of actively cycling cells) and longer cell cycle duration, resulting in prolonged existence of the short term repopulating cells in vivo. Finally, there was decreased cellular output from both individual epidermal stem cells and transit amplifying cells with aging (> Fig. 19.1). This suggests that increased cell cycle
. Figure 19.1 Epidermal repopulating units from aged progenitors are smaller and contain less cells than those from young progenitors at 1 and 11 weeks (bar = 10 mm)
Aging of Epidermal Stem Cells
duration contributes to the decreased cellular output from epidermal progenitors, while the larger growth fraction may be a compensatory mechanism [78].
5. 6.
Aging, Carcinogenesis, and Stem Cells Given the ability of stem cells to self-renew, proliferate and maintain homeostasis and the increase in incidence of cancer with aging, it is assumed that stem cells are involved in carcinogenesis. Recently a mechanistic link has been made between aging and carcinogenesis. It has been shown that senescent cells are more resistant to apoptosis [79] and have impaired DNA repair mechanisms [80]. Furthermore, senescent cells can survive for long periods of time, leading to the accumulation of damaged cells [81]. The resulting genetic instability leads to an increase in carcinogenesis (for review see [82]). The cancer stem cell hypothesis is being verified in different tissues including skin (for review see [83]). Because of similarities in the behavior of aging stem cells, senescent cells and cancer stem cells, it is thought that aged stem cells have a major role in the increased carcinogenesis associated with aging (for review see [84]).
7. 8.
9. 10. 11. 12. 13. 14.
15. 16.
17.
Conclusion This review discusses the changes in progenitor populations that occur with aging, and more specifically changes of the epidermis that occur with aging. The consensus of opinion appears to be that changes responsible for aging of tissues occur not only in the stem cell pool itself, but also in the transit amplifying cell compartment and in the stem cell environment. In order to study aging of epidermal stem cells it is essential to isolate epidermal stem cells at the single cell level to better define them at a molecular level. It will also be important to study the intrinsic and extrinsic changes that occur in the environment/niche of the epidermal stem cell with aging.
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30. Taylor G, et al. Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell. 2000;102:451–461. 31. Oshima H, et al. Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell. 2001;104:233–245. 32. Ghadially R. In search of the elusive epidermal stem cell. In: The Promises and Challenges of Regenerative Medicine. Heidelberg: Springer, 2005. p. 45–62. 33. Schneider TE, et al. Measuring stem cell frequency in epidermis: a quantitative in vivo functional assay for long-term repopulating cells. Proc Natl Acad Sci USA. 2003;100:11412–11417. 34. Kaur P. Interfollicular epidermal stem cells: identification, challenges, potential. J Invest Dermatol. 2006;126:1450–1458. 35. Nishimura EK, et al. Dominant role of the niche in melanocyte stemcell fate determination. Nature. 2002;416:854–860. 36. Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity. 1994;1:661–673. 37. Terunuma A, et al. Side population keratinocytes resembling bone marrow side population stem cells are distinct from labelretaining keratinocyte stem cells. J Invest Dermatol. 2003;121: 1095–1103. 38. Triel C, et al. Side population cells in human and mouse epidermis lack stem cell characteristics. Exp Cell Res. 2004;295:79–90. 39. Li A, Simmons PJ, Kaur P. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci USA. 1998;95:3902–3907. 40. Terunuma A, et al. Stem cell activity of human side population and alpha6 integrin-bright keratinocytes defined by a quantitative in vivo assay. Stem Cells. 2007;25:664–669. 41. Strachan LR, et al. Rapid adhesion to collagen isolates murine keratinocytes with limited long-term repopulating ability in vivo despite high clonogenicity in vitro. Stem Cells. 2008;26:235–243. 42. Bickenbach JR. Identification and behavior of label-retaining cells in oral mucosa and skin. J Dent Res. 1981;60 (Spec No C):1611–1620. 43. Yang A, et al. P63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature. 1999;398:714–718. 44. Stasiak PC, et al. Keratin 19: predicted amino acid sequence and broad tissue distribution suggest it evolved from keratinocyte keratins. J Invest Dermatol. 1989;92:707–716. 45. Lyle S, et al. The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells. J Cell Sci. 1998;111(Pt 21):3179–3188. 46. Zhu AJ, Watt FM. Beta-catenin signalling modulates proliferative potential of human epidermal keratinocytes independently of intercellular adhesion. Development. 1999;126:2285–2298. 47. Lin KK, Goodell MA. Purification of hematopoietic stem cells using the side population. Methods Enzymol. 2006;420:255–264. 48. Uezumi A, et al. Functional heterogeneity of side population cells in skeletal muscle. Biochem Biophys Res Commun. 2006;341:864–873. 49. Kohno K, et al. The direct activation of human multidrug resistance gene (MDR1) by anticancer agents. Biochem Biophys Res Commun. 1989;165:1415–1421. 50. Sleeman MA, Watson JD, Murison JG. Neonatal murine epidermal cells express a functional multidrug-resistant pump. J Invest Dermatol. 2000;115:19–23. 51. Dunnwald M, et al. Isolating a pure population of epidermal stem cells for use in tissue engineering. Exp Dermatol. 2001;10:45–54. 52. Yano S, et al. Characterization and localization of side population cells in mouse skin. Stem Cells. 2005;23:834–841.
53. Tani H, Morris RJ, Kaur P. Enrichment for murine keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci USA. 2000; 97:10960–10965. 54. Kim DS, et al. Isolation of human epidermal stem cells by adherence and the reconstruction of skin equivalents. Cell Mol Life Sci. 2004; 61:2774–2781. 55. Watt FM. Studies with cultured human epidermal keratinocytes: potential relevance to corneal wound healing. Eye. 1994;8(Pt 2): 161–162. 56. Jones PH, Harper S, Watt FM. Stem cell patterning and fate in human epidermis. Cell. 1995;80:83–93. 57. Kaur P, et al. Keratinocyte stem cell assays: an evolving science. J Investig Dermatol Symp Proc. 2004;9:238–247. 58. Kolodka TM, Garlick JA, Taichman LB. Evidence for keratinocyte stem cells in vitro: long term engraftment and persistence of transgene expression from retrovirus-transduced keratinocytes. Proc Natl Acad Sci USA. 1998;95:4356–4361. 59. Cerimele D, Celleno L, Serri F. Physiological changes in ageing skin. Br J Dermatol. 1990;122(Suppl 35):13–20. 60. Gerstein AD, et al. Wound healing and aging. Dermatol Clin. 1993;11:749–757. 61. Gilchrest BA. In vitro assessment of keratinocyte aging. J Invest Dermatol. 1983;81:184s–189s. 62. Grove GL. Age-related differences in healing of superficial skin wounds in humans. Arch Dermatol Res. 1982;272:381–385. 63. Grove GL, Kligman AM. Age-associated changes in human epidermal cell renewal. J Gerontol. 1983;38:137–142. 64. Haratake A, et al. Intrinsically aged epidermis displays diminished UVB-induced alterations in barrier function associated with decreased proliferation. J Invest Dermatol. 1997;108:319–323. 65. Leyden JJ, et al. Age-related differences in the rate of desquamation of skin surface cells [proceedings]. Adv Exp Med Biol. 1978;97: 297–298. 66. Roberts D, Marks R. The determination of regional and age variations in the rate of desquamation: a comparison of four techniques. J Invest Dermatol. 1980;74:13–16. 67. Ito M, et al. Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat Med. 2005;11: 1351–1354. 69. Nouy PLD. Biological Time. New York: The Macmillan Company, 1937. 69. Rheinwald JG, Green H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature. 1977;265:421–424. 70. Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell. 1975;6:331–343. 71. Barrandon Y, Green H. Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA. 1987;84:2302–2306. 72. Dunnwald M, et al. Mouse epidermal stem cells proceed through the cell cycle. J Cell Physiol. 2003;195:194–201. 73. Youn SW, et al. Cellular senescence induced loss of stem cell proportion in the skin in vitro. J Dermatol Sci. 2004;35:113–123. 74. Liang L, et al. As epidermal stem cells age they do not substantially change their characteristics. J Investig Dermatol Symp Proc. 2004;9:229–237. 75. Giangreco A, et al. Epidermal stem cells are retained in vivo throughout skin aging. Aging Cell. 2008;7:250–259.
Aging of Epidermal Stem Cells 76. Harrison DE, Astle CM, Stone M. Numbers and functions of transplantable primitive immunohematopoietic stem cells. Effects of age. J Immunol. 1989;142:3833–3840. 77. Sudo K, et al. Age-associated characteristics of murine hematopoietic stem cells. J Exp Med. 2000;192:1273–1280. 78. Charruyer A, et al. Transit-amplifying cell frequency and cell cycle kinetics are altered in aged epidermis. J Invest Dermatol. 2009; 129(11):2574–2583. 79. Gniadecki R, Hansen M, Wulf HC. Resistance of senescent keratinocytes to UV-induced apoptosis. Cell Mol Biol (Noisy-le-grand). 2000;46:121–127. 80. Matta JL, et al. DNA repair and nonmelanoma skin cancer in Puerto Rican populations. J Am Acad Dermatol. 2003;49:433–439.
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50 Aging of Skin Cells in Culture Suresh I. S. Rattan
Introduction The study of age-related changes in the physiology, biochemistry, and molecular biology of isolated skin cell populations in culture has greatly expanded the understanding of the fundamental aspects of skin aging. In modern biogerontology, the terms ‘‘cellular aging,’’ ‘‘cell senescence,’’ or ‘‘replicative senescence’’ most commonly imply the study of normal diploid cells in culture, which during serial subcultivation undergo a multitude of changes culminating in the permanent cessation of cell division. This process of cellular aging in vitro is generally known as the Hayflick phenomenon, and the limited division potential of normal cells is called the Hayflick limit, in recognition of the observations first reported by Leonard Hayflick in 1961 [1]. With respect to skin aging, three main cell types have been studied extensively with respect to cellular aging in vitro: dermal fibroblasts, epidermal keratinocytes, and melanocytes [2–7]. The aim of this chapter is to describe the experimental system of aging of skin cells in culture, to provide an overview of the age-related changes in the structural and functional aspects of cells including physiological, biochemical, and molecular changes, and to evaluate the use of such a system in testing and developing effective interventions for maintaining and/or re-achieving a healthy skin during aging.
Experimental Model System of Cellular Aging in Culture Once the primary culture of normal cells is established in culture from the normal tissue (e.g., a skin biopsy), by using any of the standard methods such as the explant growth and enzymic dissociation of cells, the primary culture can then be subcultivated repeatedly at each time it becomes confluent. This repeated subculturing of cells is also known as serial passaging [1]. In a description of the Hayflick phenomenon, Phase I is the period of the establishment of the primary culture from normal tissue; Phase II is a relatively long period of serial passaging,
growth, and cell proliferation at a constant rate; and Phase III, is the final period of slowing-down of growth, which results in the cessation of cell division and end of replicative lifespan of cells. The whole duration of serial passaging is considered as the process of cellular aging and the end-stage irreversible growth arrest in G1 is termed as replicative senescence. After reaching a state of replicative senescence, some cells can still stay alive and be metabolically active at a minimal level for sometime and generally resist undergoing apoptosis [1]. Although the exact culturing conditions, such as the type of the culture medium, the source of growth factors, the use of antibiotics, and the incubation temperature, humidity, and gaseous composition may vary for different cell types, serial subcultivation of normal diploid cells can be performed only a limited number of times. This is in contrast to the high proliferative capacity of transformed, cancerous, and immortalized cells, whose cultures can be subcultivated and maintained indefinitely. The total number of cell divisions, measured as the cumulative population doublings (CPD), which can be achieved by a specific cell type in vitro, depends upon several biological factors. These include the maximum lifespan of the species, developmental and adult age of the donor of the tissue biopsy, the site of the biopsy, and the health status of the donor [8]. For example, for human fibroblasts the range of CPD for the cell strains originating from embryonic tissues is between 50 and 70, whereas for those originating from adult biopsies it is generally less than 50 CPD. A similar range for CPD attained by human keratinocytes and melanocytes has been reported [2–7]. Additionally, gaseous composition, especially oxygen levels, and the quality of the nutritional serum and growth factors added to the culture medium, can significantly affect the proliferative lifespan of cells in vitro. For example, culturing of fibroblasts in vitro in the air with about 20% oxygen levels reduces their replicative lifespan, which could be otherwise achieved at low level (2%) concentration akin to in vivo conditions [9, 10]. Furthermore, the site of the skin biopsy, for example, sun-exposed versus sun-protected area has a significant effect on the CPD levels achieved by cells in culture [1, 11, 12].
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_50, # Springer-Verlag Berlin Heidelberg 2010
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The Phenotype of Aging Skin Cells Serial passaging of normal diploid skin cells is accompanied by a progressive and accumulative occurrence of a wide variety of changes before the final cessation of cell replication occurs. The emerging senescent phenotype of serially passaged normal diploid skin cells can be categorized into the structural, physiological, and biochemical and molecular phenotypes, which can be used as biomarkers of cellular aging in vitro, as summarized in > Tables 50.1–50.3. There are more than 200 such structural, physiological, biochemical, and molecular characteristics that have been studied during cellular aging, and a list of major characteristics that appear progressively in cell cultures, and distinguish between young and senescent cells, generally before the end of proliferative lifespan and their irreversible arrest in the G1 phase of the cell cycle, can be found in several publications [13–15]. A summary of such phenotypic changes is given below. . Table 50.1 Structural phenotype of skin cells undergoing aging in culture ● Increased cell size ● Change of shape from thin, long, and spindle-like to flattened and irregular ● Loss of fingerprint-like arrangement in parallel arrays on the cell culture substrate ● Rod-like polymerization of the cytoskeletal actin filaments and disorganized microtubules ● Increased membrane rigidity ● Increased multinucleation ● Increased number of vacuoles and dense lysosomal autophagous bodies
Structural phenotype > Table 50.1 lists the major structural changes observed in aging skin cells in culture. Most commonly, a progressive increase in cell size and the loss of homogenous morphological pattern are the most dramatic and easily identifiable differences in early passage young and late passage old or senescent cells. Other structural changes during aging of skin cells include cytoskeletal and membrane rigidity, accumulation of intracellular debris, and incomplete cytokinesis leading to multinucleation (> Fig. 50.1). In addition to the gross structural alterations listed in > Table 50.1, there are several ultrastructural changes reported by using electron microscopic methods. These include the presence of distorted mitochondria, increased level of chromosomal aberrations, overcondensation of chromatin, increased nucleolar fragmentation, and the accumulation of lipid–protein conjugate lipofuscin in lysosomes [16–18]. Functional phenotype Numerous studies have been performed elucidating changes in various functional and physiological parameters of skin cells undergoing aging. > Table 50.2 lists some of the main changes, which clearly indicate that almost all aspects of cellular function and physiology become impaired during aging. Collectively, these data show that aging skin cells progressively become less active, have reduced ability to maintain various physiological functions, and become more prone to the negative effects of harmful substances.
. Table 50.3 Biochemical and molecular phenotype of skin cells undergoing aging in culture ● Permanent growth arrest in late G1 phase of the cell cycle near the S phase boundary ● Increased mRNA and protein levels of cell cycle inhibitors
. Table 50.2 Physiological phenotype of skin cells undergoing aging in culture ● Altered calcium flux, pH, viscosity, and membrane potential
● Increased mRNA and protein levels of inhibitors of proteases ● Decreased expression, levels, and activities of numerous house-keeping enzymes ● Decreased expression, levels, and activities of macromolecular turnover pathways ● Reduced levels of methylated cytosines in the DNA
● Reduced activity of ionic pumps
● Reduced length of telomeres
● Reduced mobility ● Reduced respiration and energy production
● Increased levels of damage in nuclear and mitochondrial DNAs
● Reduced response to growth factors and other mitogens
● Increased levels of damaged and abnormal proteins
● Increased sensitivity to toxins, drugs, irradiation, and other stresses
● Increased levels of macromolecular cross-linking ● Increased levels of reactive oxygen species
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. Figure 50.1 This figure shows Giemsa-stained light microscopic phase-contrast pictures of serially passaged human skin fibroblasts at various points in their in vitro lifespan. Sparse and confluent cultures at three stages during replicative lifespan are compared: (1) early passage young adult skin fibroblasts with less than 30% lifespan completed; (2) middle-aged cells with 60–80% replicative lifespan completed; and (3) late passage senescent cells with more than 95% lifespan completed
Altered responsiveness of cells during aging is one of the most significant age-related changes, which can be a rate-limiting factor for the use of any potential modulators of aging. Several studies have been performed in order to understand the mechanisms for age-related alteration of responsiveness, and the pathways include unaltered receptor numbers and affinities, ineffective signal transduction, and interrupted networks [8]. Biochemical and molecular phenotype At the biochemical and molecular levels, a large body of data is available, which indicates that skin cells undergo a plethora of changes, which form the mechanistic bases of structural and physiological alterations. > Table 50.3 gives a list of main categories of biochemical and molecular changes that have been reported in aging skin cells in culture.
Depending on the available technologies and the prevailing trends, changes in the amounts and activities of thousands of proteins, and in the levels of thousands of mRNAs have been reported for aging skin cells. Recently, data are beginning to be collected for age-related changes in the so-called epigenome, metabolome, and proteome, including posttranslational modifications [19–21]. All such data will further strengthen the descriptive understanding of the phenomenon of aging of skin cells. Although every single piece of descriptive data for aging skin cells is yet to be collected, a generalized picture of the aging phenomenon has emerged. Therefore, based on the large amount of data collected so far, important inferences and generalizations can already be made, which
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have implications with respect to developing effective interventions for a healthy skin. These are as follows: 1. The rate and phenotype of aging is different in different cell types. There are both common features and specific features of aging of skin fibroblasts, keratinocytes, melanocytes, and other cell types. 2. A progressive accumulation of damage in all types of macromolecules is a universal feature of cellular aging in all cell types. 3. Replicative senescence of cells in culture is not due to the activation of any aging-specific genes, but is an indirect consequence of occurrence and accumulation of molecular damage and molecular heterogeneity. 4. A progressive failure of molecular maintenance and repair pathways is the ultimate cause of cellular aging.
From Cellular Aging In Vitro to Understanding Aging In Vivo The Hayflick system of aging of skin cells in culture has proved to be very useful in developing the cellular and molecular understanding of the overall process of aging. A loss of proliferative capacity of any of the cell types has a deteriorative impact on the functioning and survival of the entire organism. A loss or slowing-down of proliferation of osteoblasts, glial cells, myoblasts, epithelial cells, lymphocytes, and fibroblasts can lead to the onset of many age-related diseases and impairments including osteoporosis, arthritis, immune deficiency, altered drug clearance, delayed wound healing, and altered functioning of the brain. Furthermore, occurrence of fully senescent or near-senescent heterogenous cells in vivo can promote dysfunctioning of the other tissues by producing harmful signals, and can also promote and stimulate the growth of other precancerous and cancerous cells [22–25]. However, the existence of the Hayflick-type senescent cells in vivo is not very well established so far. A commonly used biomarker of senescent cells is the so-called senescenceassociated beta-galactosidase (SABG), which has been used to demonstrate the presence of senescent cells in human skin and some other tissues [23, 26, 27]. However, there are several limitations regarding the use of SABG as a marker of cellular aging in vitro, since SABG can also be detected in immortal cells under various conditions [28]. More and multiple independent markers of senescent cells are needed for this purpose. The correlation between cellular aging in vitro and in vivo is often based on the evidence gathered from studies on the effects of donor age, species lifespan, and
premature aging syndromes on cellular proliferative capacity in culture. These studies indicate that the genetic and intrinsic Hayflick limit of diploid cell strains in culture is a true reflection of what is going on during aging of an organism. However, there are some recent critiques of this based on the replicative potential of stem cells, which in the case of the skin appear to be maintained throughout lifespan [8, 29, 30]. In contrast to this, there is evidence showing that the stem cell population in the skin also undergoes aging, and the number of stem cells declines as a function of donor age and during aging of the skin equivalents in vitro [31].
Modulators of Aging Skin Cells The Hayflick system of cellular aging in culture is primarily a model for the study of slow and progressive accumulation of damage resulting in the arrest of cells in a nonproliferative state [1]. This system has been proved to be very useful for testing various physical, chemical, and biological conditions for their harmful or beneficial effects, and for understanding other aspects of cellular aging with implications in the origin of age-related diseases. For example, irradiation, severe oxidative stress by UV, hydrogen peroxide, or dicarbonyls, and gene transfection have been used to induce a sudden and rapid increase in molecular damage, resulting in premature appearance of the senescent phenotype [32–34]. On the other hand, insertion of catalytically active component of the telomerase gene can completely bypass the Hayflick limit in many cell types including skin cells, and such cells can proliferate indefinitely with or without becoming transformed [35, 36]. Similarly, normal diploid cells can be transformed and immortalized by chemical carcinogens, irradiation, and viral genes. Such approaches are helpful for unravelling the molecular details of cell cycle regulation in normal cells and its dysregulation in cancer cells [36]. The Hayflick system of cellular aging in culture has also been very useful for testing various natural and synthetic molecules as potential anti-aging compounds for the skin. Some of the well-tested examples are cytokinins kinetin and zeatin [37, 38], a dipeptide carnosine [39, 40], and extracts from medicinal plants and some algae [41]. Several of these tests have resulted in the successful development, production, and marketing of various products with pharmaceutical, cosmeceutical, and nutritional applications [42–45]. Another use of the model system of cellular aging in culture has been to test the principle of mild
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stress-induced beneficial and anti-aging effects, which is the phenomenon of hormesis [46]. For example, human skin fibroblasts and keratinocytes exposed to repeated mild heat stress (41 C, 1 h, twice a week) show several hormetic effects, such as improved protein degradation pathways, higher levels of chaperones, increased resistance to other stresses, improved differentiation, and increased proliferative lifespan [7, 46–48]. Such studies can form the basis of testing novel hormetic agents, including potential hormetins of natural or synthetic origin, for improved skin care during aging [7, 46–48].
Conclusion In conclusion, it may be reemphasized that the present understanding of the cellular and molecular basis of aging of the skin owes a lot to the use of the Hayflick system of aging of skin cells in culture. Most importantly, studies performed by using this model system have demonstrated that aging of cells is characterized by the accumulation of damage in various molecules, which results in the failure of maintenance and repair systems. Detailed genomic, proteomic, and metabolomic studies using this system can further identify the interacting networks of regulatory pathways, which may be accessible to modulation for the maintenance of the structural and functional integrity of the skin.
Cross-references > The
Use of Reconstructed Skin to Create New In Vitro Models of Skin Aging with Special Emphasis on the Flexibility of Reconstructed Skin
References 1. Rattan SIS. Cellular senescence in vitro. In: Encyclopedia of Life Sciences. 2008. doi: 10.1002/9780470015902.a0002567.pub2. 2. Norsgaard H, Clark BFC, Rattan SIS. Distinction between differentiation and senescence and the absence of increased apoptosis in human keratinocytes undergoing cellular aging in vitro. Exp Gerontol. 1996;31:563–570. 3. Yaar M, Gilchrest BA. Ageing and photoageing of keratinocytes and melanocytes. Clin Exp Dermatol. 2001;26:583–591. 4. Lin JY, Fisher DE. Melanocyte biology and skin pigmentation. Nature. 2007;445:843–850. 5. Berge U, Behrens J, Rattan SIS. Sugar-induced premature aging and altered differentiation in human epidermal keratinocytes. Ann N Y Acad Sci. 2007;1100:524–529. 6. Berge U, Kristensen P, Rattan SIS. Kinetin-induced differentiation of normal human keratinocytes undergoing aging in vitro. Ann N Y Acad Sci. 2006;1067:332–336.
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7. Berge U, Kristensen P, Rattan SIS. Hormetic modulation of differentiation of normal human epidermal keratinocytes undergoing replicative senescence in vitro. Exp Gerontol. 2008;43:658–662. 8. Cristofalo VJ, Lorenzini A, Allen RG, Torres C, Tresini M. Replicative senescence: a critical review. Mech Age Dev. 2004;125:827–848. 9. Packer L, Fuehr K. Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature. 1977;267:423–425. 10. Chen Q, Fischer A, Reagan JD, Yan L-J, Ames BN. Oxidative DNA damage and senescence of human diploid fibroblast cells. Proc Natl Acad Sci USA. 1995;92:4337–4341. 11. Pinnell SR. Cutaneous photodamage, oxidative stress, and topical antioxidant protection. J Am Acad Dermatol. 2003;48:1–19. 12. Knott A, et al. Deregulation of versican and elastin binding protein in solar elastosis. Biogerontology. 2009;10:181–190. 13. Holliday R. Understanding Ageing. Cambridge: Cambridge University Press, 1995, pp. 207. 14. Rattan SIS. Ageing – a biological perspective. Molec Aspects Med. 1995;16:439–508. 15. Marcotte R, Wang E. Replicative senescence revisited. J Gerontol Biol Sci. 2002;57A:B257–B269. 16. Macieira-Coelho A. Ups and downs of aging studies in vitro: the crooked path of science. Gerontology. 2000;46:55–63. 17. Lezhava T. Human chromosomes and aging: from 80 to 114 years. New York: Nova Sciience Publishers, 2006. 18. Stroikin Y, Dalen H, Brunk UT, Terman A. Testing the ‘‘garbage’’ accumulation theory of aging. mitotic acitivity protects cells from death induced by inhibition of autophagy. Biogerontology. 2005; 6:39–47. 19. Borlon C, et al. Expression profiling of senescent-associated genes in human dermis from young and old donors. Proof-of-concept study. Biogerontology. 2008;9:197–208. 20. Molinari J, Ruszova E, Velebny V, Robert L. Effect of advanced glycation endproducts on gene expression profiles of human dermal fibroblasts. Biogerontology. 2008;9:177–182. 21. Xie L, Pandey R, Xu B, Tsaprailis G, Chen QM. Genomic and proteomic profiling of oxdiative stress response in human diploid fibroblasts. Biogerontology. 2009;10:125–151. 22. Krtolica A, Parrinello S, Lockett S, Desprez P-Y, Campisi J. Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proc Natl Acad Sci USA. 2001;98: 12072–12077. 23. Parrinello S, Coppe JP, Krtolica A, Campisi J. Stromal-epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation. J Cell Sci. 2005;118:485–496. 24. Campisi J. Senescent cells, tumor suppression, and orgnismal aging: good citizens, bad neighbors. Cell. 2005;120:513–522. 25. Blagosklonny MV, Campisi J. Cancer and aging: more puzzles, more promises? Cell Cycle. 2008;7:2615–2618. 26. Dimri GP, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA. 1995;92:9363–9367. 27. Hornsby PJ. Cellular senescence and tissue aging in vivo. J Gerontol Biol Sci. 2002;57A:B251–B256. 28. Yang NC, Hu ML. The limitations and validities of senescence associated-b-galactosidase activity as an aging marker for human foreskin fibroblast Hs68 cells. Exp Gerontol. 2005;40:813–819. 29. Rubin H. The disparity between human cell senescence in vitro and lifelong replication in vivo. Nat Biotechnol. 2002;20:675–681. 30. Giangreco A, Qin M, Pintar JE, Watt FM. Epidermal stem cells are retained in vivo throughout skin aging. Aging Cell. 2008; 7:250–259.
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31. Youn SW, et al. Cellular senescence induced loss of stem cell proportion in the skin in vitro. J Dermatol Sci. 2005;35:113–123. 32. Toussaint O, et al. Stress-induced premature senescence as alternative toxicological method for testing the long-term effects of molecules under development in the industry. Biogerontology. 2000;1:179–183. 33. Sejersen H, Rattan SIS. Dicarbonyl-induced accelerated aging in vitro in human skin fibroblasts. Biogerontology. 2009;10:203–211. 34. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell. 1997;88:593–602. 35. Simonsen JL, et al. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells. Nat Biotechnol. 2002;20:592–596. 36. Collado M, Blasco MA, Serrano M. Cellular senescence in cancer and aging. Cell. 2007;130:223–233. 37. Rattan SIS, Clark BFC. Kinetin delays the onset of ageing characteristics in human fibroblasts. Biochem Biophys Res Commun. 1994;201:665–672. 38. Rattan SIS, Sodagam L. Gerontomodulatory and youth-preserving effects of zeatin on human skin fibroblasts undergoing aging in vitro. Rejuven Res. 2005;8:46–57. 39. McFarland GA, Holliday R. Retardation of the senescence of cultured human diploid fibroblasts by carnosine. Exp Cell Res. 1994;212:167–175.
40. McFarland GA, Holliday R. Further evidence for the rejuvenating effects of the dipeptide L-carnosine on cultured human diploid fibroblasts. Exp Gerontol. 1999;34:35–45. 41. Nizard C, et al. Algae extract protection effect on oxidized protein level in human stratum corneum. Ann N Y Acad Sci. 2004; 1019:219–222. 42. Glaser DA. Anti-aging products and cosmeceuticals. Facial Plast Surg Clin N Am. 2004;12:363–372. 43. Rattan SIS. N6-furfuryladenine (kinetin) as a potential anti-aging molecule. J Anti-Aging Med. 2002;5:113–116. 44. Chiu PC, Chan CC, Lin HM, Chiu HC. The clinical anti-aging effects of topical kinetin and niacinamide in Asians: a randomized, double-blind, placebo-controlled, split-face comparative trial. J Cosmet Dermatol. 2007;6:247–253. 45. McCullough JL, Weinstein GD. Clinical study of safety and efficacy of using topical kinetin 0.1% (Kinerase) to treat photodamaged skin. Cosmet Dermatol. 2002;15:29–32. 46. Rattan SIS. Hormesis in aging. Ageing Res Rev. 2008;7:63–78. 47. Rattan SIS, Ali RE. Hormetic prevention of molecular damage during cellular aging of human skin fibroblasts and keratinocytes. Ann N Y Acad Sci. 2007;1100:424–430. 48. Rattan SIS. Hormetic modulation of aging in human cells. In: Le Bourg E, Rattan SIS (eds) Mild Stress and Healthy Aging: Applying Hormesis in Aging Research and Interventions. Dordrecht: Springer, 2008, pp. 81–96.
Metabolism
29 Alterations of Energy Metabolism in Cutaneous Aging Thomas Blatt . Horst Wenck . Klaus-Peter Wittern
Introduction Aging is understood as the result of a complex interaction of biological processes that are caused by both environmental processes (extrinsic aging) and genetic processes (intrinsic aging). Research into the biology of aging has provided detailed insight into the molecular mechanisms of age-related changes in organs, tissues, and cells. Most information relating to intrinsic aging processes comes from tissues other than the skin. This is in part due to the fact that clinically manifest diseases such as Type2 diabetes or neurodegenerative disease are often correlated with aging of cells. In part it is also due to the fact that substantial amounts of primary cells and organelles for biochemical analyses can be more easily isolated from other organs such as muscle, brain, or liver, as compared with skin. Nevertheless, intrinsic aging is based on general biological processes that apply more or less to all proliferating cells and terminally differentiated cells as well. Therefore, general intrinsic aging processes seen in a liver cell, muscle cell, or neuron can be expected also to apply more or less to skin cells. In fact, most of the aging processes identified and studied with other cells could also be confirmed with keratinocytes or dermal fibroblasts, even though some downstream details may be different. Extrinsic aging processes have been intensively studied in the skin though. This applies especially to a process called photoaging, which is induced by the skin’s most dominant stressor – UV light. In contrast to skin, UV light is an irrelevant stressor to other tissues. Researchers have developed a battery of slightly invasive or non-invasive biophysical measurement procedures to study UV-induced stress and aging-related damage in situ even in small skin samples. Furthermore, many in vitro methods are available to study photoaging based on cultured cells or threedimensional cultured skin models. Besides UV light, there are many other extrinsic stressors, encompassing, for example, environmental chemicals, nutritional conditions, or even hormonal imbalances which may induce extrinsic aging of the skin and other organs as well.
This overview will focus on aspects of energy metabolism in cutaneous aging. These aspects are especially important since human skin tissue, being exposed to a plethora of endogenous and environmental stress factors, is highly dependent on energy supply in order to combat cellular deregulation and/or to repair damage. As detailed below, cellular energy levels decline during intrinsic and extrinsic aging as well, and consequently the capacity of the skin to counteract environmental stress declines with aging. Decreased compensation of environmental stress and insufficient repair, in turn, accelerate skin aging, which consequently leads to further decline of cellular energy levels in the skin. Breaking this feedback loop by sustaining cellular energy levels in the skin is thought to decelerate, stop, or even reverse intrinsic and extrinsic skin aging, with compounded interest over time.
Phenomenology of Energetic Factors Associated with Skin Aging The energy demand of skin cells is supplied by two major primary sources – mitochondrial oxidative phosphorylation and glycolysis. In addition to these major primary sources, skin cells may also utilize a major secondary energy store for situations of acute high energy demand – the creatine/phosphocreatine system. As detailed below, all three major energy sources are affected by intrinsic and extrinsic skin aging and offer potential entry points for intervention strategies to decelerate the aging process.
Aging Effects on Mitochondrial Function The mitochondria, as small oxidative power-plants in the cells, play a pivotal role in energy supply. Beyond energy production, mitochondria can perform other pivotal cellular functions such as regulation of programmed cellular death (apoptosis). These organelles contain their own genetic material, mitochondrial DNA (mtDNA), which
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_29, # Springer-Verlag Berlin Heidelberg 2010
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is maternally inherited. Although much smaller than the nuclear genome, mtDNA is equally important, as it has been shown to play a crucial role in aging, as discussed in detail in the section Genetic Damage to Mitochondria of this overview. This central organelle of energy metabolism and control of cell death is supposed to be a target for aging and a promoter of aging as well [1]. Within living cells, mitochondria are observed as small sausage-shaped organelles, longer snake-like tubules, branched reticula, extended filaments and networks or clusters that are connected via intermitochondrial junctions. Mitochondrial morphology is regulated in many cultured eukaryotic cells by fusion and fission, and a tightly controlled balance between fission and fusion events is supposed to ensure normal mitochondrial ultrastructure as well as mitochondrial and cellular functions. Mitochondria of old endothelial cells show a significant and equal decrease of both fusion and fission activity [2], indicating that these processes are sensitive to aging and are likely to contribute to the accumulation of damaged mitochondria during aging [3]. Aging of cells is also associated with aberrations of mitochondrial morphology and ultrastructure. Typical changes of mitochondrial ultrastructure during aging, as can be seen by electron microscopy in cultured fibroblasts, are loss of branched mitochondria in old cells, enlargement of mitochondria, matrix vacuolization, shortened cristae, and loss of dense granules [4]. Furthermore, cystic blebs are evident in mitochondria of some cells with an apparent increase in old cells. These blebs appear to be due to weakening of the inner membrane, allowing dilatation of the outer membrane which otherwise appears intact. Similar ultrastructural changes of mitochondria as seen in aged cells are also seen in cultivated skin fibroblasts of patients with point mutations in mitochondrial DNA (mtDNA) affecting the energy metabolism of the organelles. The changes encompass partially swollen mitochondria with unusual and sparse cristae, heterogeneity of cristae in size and shapes or their absence, as well as almost complete absence of branched mitochondria [5]. Furthermore, similar changes in mitochondrial ultrastructure as seen in aged fibroblasts are also seen in photoaged keratinocytes chronically exposed to low doses of UV-B irradiation [6]. Thus, ultrastructural changes of mitochondria in intrinsically aged cells are similar to those seen in mitochondria from skin cells damaged by intrinsic genetic defects or damaged by exogenous stressors leading to photoaging. Since morphological features typical of mitochondria from aged cells can also be induced in mitochondria from otherwise normal cultured primary skin fibroblasts by inhibition of energy
metabolism with drugs targeting the respiratory chain [7], changes of the mitochondrial ultrastructure can be hypothesized to be a cause and consequence of altered mitochondrial energy metabolism as well. The central function of the mitochondrial network in the cell is the production of Adenosintriphosphate (ATP) by transfer of electrons from digested food to carrier molecules such as Nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD+), thus generating NADH and FADH, and subsequent delivery of the electrons to the respiratory Oxidative phosphorylation generates mitochondrial ATP by means of five multiple subunit enzyme complexes (I through V) plus the adenine nucleotide translocator (ANT), all localized within the mitochondrial inner membrane. Complexes I–IV constitute the electron transport chain. Reduced NADH is oxidized by Complex I (NADH dehydrogenase), and succinate is oxidized by Complex II (succinate dehydrogenase); the electrons are transferred to ubiquinone (Coenzyme Q10) to yield ubiquinol. The electrons from ubiquinol are transferred to Complex III (ubiquinol:cytochrome c oxidoreductase), then to cytochrome c, then to Complex IV (cytochrome c oxidase), and finally to oxygen. The energy released is used to pump protons out of the mitochondrial inner membrane through Complexes I, III, and IV, and the resulting electrochemical gradient is exploited by Complex V (ATP synthase) to condense Adenosindiphosphate (ADP) and inorganic phosphate to form ATP. Both ATP and ADP are exchanged across the mitochondrial inner membrane by ANT. The five protein complexes of the electron transport chain work as an integrated system, with mitochondrial DNA (mtDNA) encoding 13 of the proteins and nuclear DNA encoding approximately 60. Formation of these complexes is a complicated procedure based on the coordinated transport and assembly of components from two different genomes and compartments [8]. As an organism ages, either by extrinsic or intrinsic aging, there is a significant decline in mitochondrial function and cellular energy balance [9, 10]. This applies especially to mitochondrial membrane potential which is key to mitochondrial function [11]. Assessment methods to monitor the mitochondrial respiration rate and membrane potential, even on a single intact cell level are discussed in the section Energetic effects of Creatine of this overview. Decrease of mitochondrial function during aging has been described as a general feature in many in vitro systems. These descriptions encompass the loss of mitochondrial membrane potential of old and postmitotic human umbilical vein endothelial cells [2], or lower
Alterations of Energy Metabolism in Cutaneous Aging
respiration rates of spleen lymphocytes isolated from from old mice as compared with lymphocytes from young mice [12], just to mention a few. In fact, the general decline of mitochondrial respiratory functions with age has also been described in human studies, for example in hepatocytes investigated in a study enrolling subjects of 31–76 years old, where a significant negative correlation between age and respiratory control and ADP/O ratios was observed [13]. Decline in skeletal muscle mitochondrial respiratory chain function has also been investigated in isolated intact skeletal muscle mitochondria in a study enrolling subjects aged 16–92 years. State 3 (activated) mitochondrial respiration rates showed a significant negative correlation between respiration rate and age. A similar trend was seen for respiratory enzyme activities assayed in muscle homogenate [14]. These findings demonstrate a substantial fall in mitochondrial function in aging muscle and liver cells, and suggest that a fall in mitochondrial oxidative capacity and membrane potential in aging cells may be an important general contributor to the aging process. Loss of mitochondrial membrane potential has also been reported in human dermal fibroblasts aged in vitro by serial passage [15]. Furthermore, a marked agingrelated decline in efficiency of oxidative phosphorylation was observed in human skin fibroblasts isolated from a large group of subjects ranging in age between 20 weeks fetal and 103 years [16]. In the latter study, the analysis of endogenous respiration rate revealed a significant decrease in the age range from 40 to 90 years, and a tendency to uncoupling in the samples from subjects above 60 years. These findings clearly pointed to a dramatic mitochondrial dysfunction, which would lead to a decrease in ATP synthesis rate in skin fibroblasts with increasing age. The impact of intrinsic aging on the mitochondrial oxidative capacity and mitochondrial membrane potential of skin cells is furthermore supported by a number of indirect studies discussed in this review, addressing the effects of aging on the generation of reactive oxygen species (ROS) in skin cells from young and old donors, or the effect of energy enhancers. Besides intrinsic aging effects on the mitochondrial capacity of skin cells, a substantial decline of the mitochondrial membrane potential can be observed following UV irradiation of keratinocytes in vitro, which is the dominant stressor leading to photoaging of cells [17, 18]. A decline of mitochondrial membrane potential after UV irradiation has also been reported in situ using suction blisters taken from irradiated and non-irradiated skin areas of healthy old volunteers with an average age of 65.2 years [17].
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Aging Effects on Anaerobic Energy Pathways The loss of mitochondrial function represents an inherent part in modern theories trying to explain the cutaneous aging process. The number of damaged mitochondria increases with aging, and as a consequence an impaired mitochondrial ATP synthesis can be observed. To assure survival of a cell, any decrease in mitochondrial energy production due to impaired mitochondrial function has to lead to compensatory actions in cellular metabolism which result in higher energy production via non-mitochondrial pathways such as glycolysis. This goes along with reports from several in vitro studies demonstrating a higher glucose uptake and lactate production at advanced cellular age in fibroblasts [19, 10]. As a speacial feature of the skin, atmospheric oxygen may be directly taken up by the human epidermis, and in theory, the flux of oxygen from the environment should be sufficient to fully cover its oxygen demand [20]. A rather surprising hypothesis, stimulated by a study from Ronquist et al. [21], is that human epidermis works to a substantial extent in an anaerobic manner. In cell culture, keratinocytes contain more lactate than do most other cell types. Their lactate production in vitro is vigorous and independent of oxygen and most of it is released to the medium. During autoincubation of the epidermis under starved conditions, energy charge values are low and comparable with those reported for smooth muscle. Moreover, the overwhelming majority of the keratinocytic mitochondria have an appearance markedly deviating from those in other cells such as Langerhans cells, melanocytes and fibroblasts, and, above all, are characterized by an enormous reduction of the inner membrane. Ronquist concludes from these findings that epidermal energy metabolism is predominantly anaerobic in spite of the formal presence of mitochondria and sufficient oxygen. According to not yet published data generated in at the authors’ institution, significant age-dependent differences in mitochondrial function can be observed in keratinocytes isolated from skin biopsies of young and old donors. The data suggest that energy metabolism shifts to a predominantly non-mitochondrial pathway and is therefore functionally anaerobic with advancing age. Primary keratinocytes derived from old donors show a higher glucose uptake compared to the cells obtained from a young donor panel, and the increased lactate production in keratinocytes from the old age group clearly indicates a suboptimal utilization of glucose and a shift in metabolism towards an increased glycolysis. The data generated so far show no differences in mitochondrial
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content and structure during aging in skin keratinocytes, indicating that the number of mitochondria does not change, but rather their function. This decline of mitochondrial function may explain the observed age-associated glycolytic activity as some kind of compensatory counterregulation. Simulation of mitochondrial dysfunction by inhibition of ATP synthase in keratinocytes from young donors leads to a comparable rise in glucose uptake and lactate production as seen in the basic, unstressed state of keratinocytes from the old age group. In fact, the energy metabolism of keratinocytes is a subject of controversy, and it is unclear why keratinocytes express a metabolic status that is, as compared with fibroblasts or endothelial cells, partially shifted to an anaerobic status. It was found that keratinocytes respire as much oxygen as fibroblasts, even though maximal activities of the respiratory chain complexes are two- to fivefold lower, whereas expression levels of respiratory chain proteins are similar. Congruent with this, superoxide anion levels are much higher in keratinocytes, and keratinocytes display higher lipid peroxidation levels and a lower reduced glutathione/oxidized glutathione ratio, indicating enhanced oxidative stress [22]. Thus, it seems that keratinocytes actively use the mitochondrial respiratory chain not only for adenosine 5’ triphosphate synthesis but also for the accumulation of superoxide anions, even at the expense of mitochondrial functional capacity. The reason for this behaviour of keratinocytes is not clear, but it may indicate that superoxide-driven processes might be a prerequisite for keratinocyte differentiation, even though the lack of energy supply via oxidative phosphorylation has to be compensated via glycolysis then.
Aging Effects on Extramitochondrial Energy Stores Precise coupling of spatially separated intracellular ATPproducing and ATP-consuming processes is fundamental to the bioenergetics of living organisms, ensuring a failsafe operation of the energetic system over a broad range of cellular functional activities, thereby securing the cellular economy and energetic homeostasis under stress [23]. Beside the mitochondrial energy supply and glycolysis as primary sources for energy, cells also have a secondary energy storage system named the creatine/phosphocreatine (Cr/PCr) pathway. This is also established in the human skin and is responsible for an extremely fast energy supply in situations of high temporal energy demand when ATP is used up faster than can be produced from primary sources [24, 25, 17], or when energy supply from
primary sources is temporarily interrupted, such as in situations of short term hypoxia or anoxia [26]. The free energy of ATP, which itself cannot be stored efficiently, is stored and transported in the form of PCr from sub-cellular sites of energy production, e.g., mitochondria, to places of high energy requirements, where creatine kinase (CK) activity can rapidly replenish cellular ATP in situ [27, 28, 29]. Predominant isoforms of CK consist of cytosolic mm-CK (muscle-type), mainly found in muscle cells, as well as cytosolic bb-CK (braintype) and ubiquitous mitochondrial mt-CK, with the last two mainly located in the brain but also present in skin [30, 31]. Creatine kinases catalyze the following reversible reaction: Mg ATP þ Cr $ PCr þ Mg ADP þ Hþ : The mt-CKs form octamers assembled from four dimers each, but only the octameric form can interact with both inner and outer mitochondrial membranes through the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC). The mt-CK activity couples the oxidative phosphorylation to mitochondrial PCr production by catalyzing the conversion of Cr to PCr at expenses of the intramitochondrially produced ATP. The PCr is exported to the cytosol, whereas the produced ADP is pumped back to the mitochondrial matrix via ANT. Cells attain their physiological levels of creatine, either by biosynthesis from the amino acids arginine, glycine, and methionine in the kidney, liver, and pancreas of vertebrates including humans, and/or alternatively by ingestion of meat and fish. Creatine is transported via the blood circulation and is taken up into cells by the Na- and Cl-dependent creatine transporter (CRT) protein [32, 33]. Once inside a cell, creatine can be stored at high concentrations (e.g., 40 mM for muscle cells). In skin, oxidative damage of cellular and extracellular components activates intrinsic repair mechanisms, which necessarily require ATP for full functionality. The PCr/CK system together with the recently discovered epidermal creatine transporter (CRT) [30] provides human skin with an important tool to cope efficiently with such conditions of high energy demand. In fact, both creatine kinase subtypes, bb-CK and mt-CK, as well as CRT, are expressed in human skin [17], showing high levels in the epidermis but less in the dermis, with the highest enzyme activity found in keratinocytes, which are generally shifted to a more anaerobic state of metabolism, as discussed in the section Aging Effects on Anaerobic Energy Pathways. Results from experiments using skeletal muscle and heart muscle clearly show that both PCr amount [34, 35, 36], and CK activity decrease with age [37, 35].
Alterations of Energy Metabolism in Cutaneous Aging
Also in skin, a reduction in the cellular concentration of creatine can be determined from the age of about 30 [38], paralleled by a slightly reduced CK activity in skin cells from older donors [17]. This decline in skin CK activity may be caused by the generation of ROS during cutaneous aging. This is supported by the fact that CK, specifically mt-CK, is a primary target for ROS, especially peroxynitrite [39]. Moreover, cutaneous cells may show signs of a declining creatine level, probably caused by a stress and age-related decline of dermal vascularization [40].
Mechanisms of Skin Aging Related to Energy Metabolism Mitochondrial Impairment by Its Own Oxidation By-products Free reactive oxygen species (ROS) are generated in the skin by several different processes [41], with exogenous stress following UV-irradiation being the most dominant generator of ROS in UV-exposed skin. Other important contributions include proteins within the plasma membrane, such as the growing family of NADPH oxidases. Furthermore, generation of H2O2 as a by-product of fatty acid degradation in peroxisomes has to be taken into account as an endogenous source of ROS. The same holds for the generation of ROS by oxidative burst of phagocytes during inflammatory reactions, as well as the activity of various cytosolic enzymes such as cyclooxygenases. Although all these sources contribute to the overall oxidative burden of a cell, the vast majority of cellular ROS (estimated at approximately 90%) which is generated in cells independently of UV-stress can be traced back to the mitochondria as by-products of impaired mitochondrial respiration [42], and oxidants generated by mitochondria are supposed to be the major source of the oxidative lesions that accumulate with age [43]. The continuous threat of oxidant damage to the cell, tissue, and organism as a whole is underscored by the existence of an impressive array of cellular defenses that have evolved to battle these reactive oxidants [44]. The cell is equipped with a variety of defence mechanisms to remove ROS. Superoxide dismutases convert superoxide into hydrogen peroxide, which in turn can be transformed into water by catalase or glutathione peroxidase. The cell also contains nonenzymatic scavengers such as ascorbate, pyruvate, flavenoids, carotenoids and glutathione which may inactivate potentially damaging ROS. However, these defenses are not perfect and, consequently, cellular macromolecules become oxidatively damaged.
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Relevant to mitochondrial function is the efficiency of electron movement through the electron transport chain and its coupling to oxidative phosphorylation to produce ATP. The coupling efficiency can be measured experimentally by determining ADP/O ratio, and by determination whether the mitochondria are in State 3 or State 4, whereby State 3 represents a condition where the rate of oxidative phosphorylation is not limited by ADP concentration, and State 4 a condition where the level of ADP limits oxidative phosphorylation. State 4 is associated with a reduced respiratory chain, leading to an ‘‘electron jam’’ and increased formation of ROS byproducts. When the rate of electron flow is slow, electrons tend to accumulate in the respiratory chain, and electrons escaping from the somewhat ‘‘leaky’’ electron transport chain (ETC) can reduce oxygen to form the highly reactive free radical superoxide anion (O2●), which, in turn, can be further reduced to hydroxyl radical (OH●) and hydrogen peroxide (H2O2). Furthermore, the superoxide anion can initiate the oxidation of sulphite or nitric oxide, resulting in ROS such as sulphur pentoxy anion or peroxynitrite. Overall, mitochondrial ROS generation is high during resting respiration, but when electrons flow quickly through the respiratory chain reducing O2 to water, the rate of ROS production is usually lower. It has been estimated that about 1012 O2 molecules are processed by each cell daily, and that the leakage of partially reduced oxygen molecules is about 2%, yielding about 2 1010 O2● and H2O2 molecules per cell per day [45]. In addition to the toxic electron transport chain reactions of the inner mitochondrial membrane, the mitochondrial outer membrane enzyme monoamine oxidase catalyzes the oxidative deamination of biogenic amines and is a quantitatively large source of H2O2 that contributes to a further increase in the steady state concentrations of reactive species within both the mitochondrial matrix and cytosol [46]. High ROS concentrations, resulting from either increased production or decreased detoxification, can cause oxidative damage to various cellular components, ultimately leading to cell death [47]. Beyond damage to mitochondria, which will be discussed in detail below, age-related features often associated with excess ROS are accumulation of oxidized intracellular proteins with age [48], the decrease of fluidity of cellular membranes with age [49], or even malfunctioning of the connective tissue remodeling process due to increased activity of extracellular matrix-degrading metalloproteinases [50], just to mention a few. Toxic reactions exerted by ROS, and imbalances in the production and removal of ROS, significantly contribute to the aging process and are the basis of the ‘‘The Free Radical Theory of Aging’’ [51], postulating that the
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production of intracellular reactive oxygen species is the major determinant of life span [42]. The hypothesis that rate of intracellular ROS production is associated with the rate of aging was tested by Sohal [52], comparing the rate of H2O2 generation by mitochondria in houseflies. The rate of mitochondrial H2O2 release was found to be associated with remaining life expectancy or the physiological age of flies. At the same chronological age, mitochondria from flies with a shorter remaining life expectancy had a markedly higher rate of H2O2 generation than those with a longer life expectancy. In another experimental model of aging – the senescenceaccelerated mouse (SAM) – animals exhibit a shortened life span (about 18 months) as compared with normal mice, as well as early manifestation of various signs of senescence including changes in physical activity, skin, and spinal curvature. In the SAM the respiratory control ratio and the ATP/O – an index of ATP synthesis – decreases more rapidly as compared with the unaffected wild-type mice. Furthermore, uncoupled respiration in liver mitochondria is markedly decreased with aging in SAM [53]. Cultured dermal fibroblasts from SAM produce more ROS within the mitochondria than do cells from wild-type control mice, coinciding with an increase in the mass of the mitochondria, degenerative mitochondrial morphology with longer culture periods, and lower membrane potential as compared with the controls [54]. Another study supporting the hypothesis that rate of intracellular ROS production is associated with the rate of aging used genetically modified mice with impaired mtDNA repair function. Secondary to the impaired mtDNA repair function all such mice exhibit a significant reduction in respiratory chain activity and ATP generation in postmitotic tissue such as heart, as well as a significantly shortened life span and the appearance of a number of age-related phenotypes, including hair loss, kyphosis, and reduced fertility [55]. These findings suggest that damaged mitochondria, which produce an excess of ROS, can accelerate the aging process in a kind of feedback loop. In fact, the mitochondria seem to be highly susceptible to harm exerted by ROS, including those produced by themselves, and increasing levels of oxidative damage in various compartments of the mitochondrium can be observed during cellular aging. Damages to mitochondrial DNA (mtDNA) – which contributes a substantial part of the proteins of the respiratory chain – isolated from rat liver or various human brain regions are at least tenfold higher than those of nuclear DNA. These higher levels of oxidative damage and mutation in mtDNA have been ascribed to location of the DNA near the inner mitochondrial
membrane sites where oxidants are formed. The extent of damage and mutation of mtDNA may be further aggravated by lack of protective histones, and lack of DNA repair activity in mitochondria. Oxidative lesions in mtDNA accumulate as a function of age, which has been well described for human diaphragm muscle [56], human brain [57]), and rat liver [58]. As in the case of oxidative damage to DNA, an ageassociated increase in oxidative damage to mitochondrial protein is also observed [59]. The accumulation of oxidized dysfunctional protein with reactive carbonyl groups can lead to inter- and intramolecular cross-links with protein amino groups and may cause loss of biochemical and physiological function in mitochondria. Thus the age-related accumulation of protein oxidation products in mitochondria may also lead to loss of energy production and increased production of oxidants. Increased oxidants may also contribute to alterations in mitochondrial membrane fluidity and phospholipid composition that occur during aging. These in turn can affect the ability of mitochondria to transport substrates and to generate sufficient proton motive force to meet cellular energy demands. With regard to lipids, part of the increased sensitivity of mitochondria to oxidants appears to be due to peculiarities in membrane lipid composition [60] which is characterized by the presence of cardiolipin. Cardiolipin serves as an insulator and stabilizes the activity of protein complexes important to the electron transport chain, and it also ‘‘glues’’ them together. Because cardiolipin plays a pivotal role in facilitating the activities of key mitochondrial inner membrane enzymes, it would be expected that changes that increase its susceptibility to oxidative damage would be deleterious to normal mitochondrial function [43]. Cardiolipin is solely synthesized in the mitochondria, and is typically present in the membranes of mitochondria, mostly in the inner membrane, which consists roughly 20% of its lipids. Cardiolipin aquires an increasing percentage of polyunsaturated fatty acids with increasing age of an organism (substitution 18:2 to 22:4 and 22:5), which renders it even more susceptible to oxidative damage. In fact, mitochondrial cardiolipin content has been reported to decrease with age in a number of tissues, including heart, liver, nonsynaptic brain mitochondria, and epithelial cells [61, 62, 63, 64], presumably due to oxidative damage. The decrease of cardiolipin with age is associated with a decrease in State 3/State 4 ratio. This loss of cardiolipin by increased susceptibility to oxidation could play a critically important role in the agerelated decrements in mitochondrial function. In addition to the established role of the mitochondria in energy metabolism, regulation of cell death has emerged
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as a second major function of these organelles. This seems also to be intimately linked to their generation of ROS [65]. Mitochondrial regulation of apoptosis occurs by mechanisms, which have been conserved through evolution, involving the release of cytochrome c into the cytoplasm which may be initiated by the oxidation of cardiolipin. Oxidation of cardiolipin, which occurs at higher rates in aged cells, reduces cytochrome c binding to mitochondrial inner membranes and increases the level of soluble cytochrome c in the intermembrane space. Subsequent release of the hemoprotein into the cytoplasm, which starts the apoptotic machinery via activation of caspases, occurs by pore formation mediated by pro-apoptotic Bcl-2 family proteins, or opening of mitochondrial permeability transition pores (MPTP). Various factors enhance the likelihood of MPTP opening, among them are dissipation of the difference in voltage between the inside and outside of mitochondrial membranes (known as permeability transition), or the presence of free radicals, both typical features of mitochondria in aged cells. ROS are also known as signaling molecules under subtoxic conditions which may activate cytoplasmic signal transduction pathways that are related to growth, differentiation, senescence, transformation and tissue degradation [66, 67, 68]. Hydrogen peroxide, for example, has been shown in different cultured cell lines to induce either apoptosis at high concentrations, or features of senescence at subtoxic concentrations [69], with cellular senescence being defined as the loss of proliferative capacity of primary cell lines, characterized by cell cycle arrest, reduced DNA synthesis, increased cell size, granularity and size heterogeneity [70, 71]. Senescent cells enter a terminally nondividing state in which they can stay for long periods before dying. Thus, in the case of stress-induced premature senescence, ROS are considered important intermediates contributing to the phenotype. The data of Zwerschke et al. [10] suggest the occurrence of significant metabolic imbalances in human fibroblasts rendered senescent by exposition to ROS. There is a drastic deregulation of the carbohydrate metabolism in senescent cells, characterized by an imbalance of glycolytic enzyme activities and the failure to maintain ATP levels. This leads to an up-regulation of adenylate kinase activity and the levels of AMP, which acts as a growth-suppressive signal that induces premature senescence. The activities of several glycolytic enzymes are strongly up-regulated. Moreover, the function of the malate–aspartate shuttle is reduced in senescent cells, preventing the transport of hydrogen into the mitochondria, where it could be used for ATP production. Instead, senescent cells activate LDH and take up pyruvate to get rid of hydrogen. Thus
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ATP-consuming steps of glycolysis are enhanced, whereas the ATP-producing steps are inhibited, and this leads to a severe reduction of the intracellular concentration of both ATP and GTP. Senescent keratinocytes and fibroblasts were also described by Campisi [72] to accumulate with age in human skin, likely due to the impact of increased endogenous oxidative stress in aged skin.
Genetic Damage to Mitochondria The mitochondrial genome is very small and economically packed, and the expression of the whole genome is essential for the maintenance of mitochondrial bioenergetic function. Thus, even small genetic alterations may have tremendous effects on mitochondrial function. In the past decade, more than 100 mtDNA mutations have been found in patients with mitochondrial disease, and some of them also occur in aging human tissues [73]. Thus, it may be hypothesized that accumulation of mitochondrial DNA deletions may be an important factor in intrinsic aging. At higher age, several independently acquired types of mtDNA mutations, accumulating clonally in certain cells, can even be found in different tissues of the same subject [74]. The incidence and abundance of mutant mtDNAs are increased with age, and much more than for nuclear DNA mutations [75, 76]. Studies of sequence heterogeneity of mitochondrial DNA from rat and mouse tissues derived from young adult and senescent animals have revealed that about 1% of the native mtDNA population in adult liver and about 5% in senescent liver having deleted/inserted segments [77]. In humans it was found that normal heart muscle and brain from adult human individuals contain low, though substantial levels of a specific mitochondrial DNA deletion, previously found only in patients affected with certain types of neuromuscular disease. This deletion was not observed in fetal heart or brain [78]. In a further human study focussing on the prevalence of mtDNA deletions in tumorous and surrounding healthy tissue, mtDNA mutations were found to be abundant in margin tissue specimens from older patients and their number correlated with the patient age. Significantly fewer deletions were detected in the tumours than the margins, and the tumours often had no deletions [79], implying a potential selection for full-length mtDNA or perhaps a protective role for mtDNA deletions in the process of tumourigenesis. Concerning photoaging, also photoaged skin is characterized at the molecular level by increased amounts of large-scale deletions of the mitochondrial genome such as
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the 4,977 bp common deletion encompassing deletion of four genes for subunits of complex I, one gene for complex IV, two genes for complex V, and several genes for mitochondrial tRNAs [80, 81]. The common deletion can be generated in dermal fibroblasts through repetitive ultraviolet UV-A irradiation [82]. For example, in a human study, previously unirradiated skin of 52 normal human individuals was repetitively exposed to physiological doses of UV-A light, and repetitive UV exposure led to an approximately 40% increase in the levels of the common deletion in skin tissue. Nine individuals were examined up to 16 mo after cessation of UV exposure and some showed accumulation up to 32-fold [83]. In another human study focussing on extrinsic photoaging, several types of mtDNA length mutations including the common 4,977 bp deletion were investigated in normal human skin tissues. It was found that the incidences of these deletions and tandem duplications of mtDNA in sun-exposed skin were all significantly higher than those in non-exposed skin [84]. Moreover, these mutations started to appear in the third decade of life, and the age at which the mutations could be detected in sun-exposed skin was substantially younger than in non-exposed skin. In another human study focussing on mtDNA aberrations in skin tissue, the frequency of a so-called T414G mutation within the control region of mtDNA, has been demonstrated to accumulate in both chronologically and photoaged skin using cultured dermal fibroblasts from donors of different age [85]. Thus, there is a strong correlation of extrinsic and intrinsic cellular aging with the accumulation of aberrations of mtDNA, and increasing evidence suggests that, due to a negative feedback loop, damaged mtDNA is a cause and consequence of aging as well. The causative role of mtDNA mutations and resulting mitochondrial dysfunction for intrinsic and extrinsic aging has been demonstrated in several experimental models and human studies. Premature aging has, for example, been reported in knock-in mice expressing a defective mitochondrial DNA polymerase. These knock-in mice develop an mtDNA mutator phenotype with a threefold to fivefold increase in the levels of point mutations, as well as increased amounts of deleted mtDNA, associated with reduced lifespan and premature onset of aging-related phenotypes such as weight loss, reduced subcutaneous fat, alopecia (hair loss), kyphosis (curvature of the spine), osteoporosis, anaemia, reduced fertility and heart enlargement [55]. The results thus provide a causative link between mtDNA mutations and aging phenotypes in mammals. It is plausible that the accumulation of mtDNA mutations leads to decreased gene expression, resulting in a decline in
oxidative phosphorylation, and inefficient electron transport, which consequently increases the generation of ROS [86]. A direct link between the amount of mtDNA aberrations and oxidative stress has been demonstrated using a series of the cybrids harboring varying proportions of mtDNA with the common 4,977 bp deletion. The population doubling time was longer for the cybrids containing higher proportions of 4,977 bp-deleted mtDNA. In addition, the respiratory function was decreased with the increase of the portion of aberrant mtDNA in the cybrids. The results also showed that the specific contents of typical cellular oxidation products stemming from ROS in cybrids harboring >65% of the aberrant mtDNA were significantly increased as compared with those of the cybrids containing undetectable mutant mtDNA [80, 87]. In a study on human fibroblasts, gradual largescale deletion of the mtDNA from unirradiated human skin fibroblasts was found to induce a gene expression profile reminiscent of photoaged skin. The modified cells exhibited an altered gene expression profile resulting from intracellular, mitochondria-derived oxidative stress, dominated by high expression of matrix metalloproteinase-1 (MMP-1) which is known to be expressed in response to oxidative stress 95]. Vice versa, the increase in ROS production is a likely promotor for additional mtDNA damage and accumulation of mtDNA mutations. The causative role of oxidative stress for the increased frequency of mitochondrial DNA aberrations has been demonstrated in a variety of experimental models and in human studies. For example, oxidative damage elicited by imbalance of free radical scavenging enzymes and its association with large-scale mtDNA deletions in aging human skin has been suggested using skin tissue derived from donors of different age. In subjects above the age of 60 years, elevated oxidative stress was caused by an imbalance between the production and removal of ROS and free radicals, and was paralleled by an increase of the proportion of mtDNA with the 4,977 bp deletion [88]. The causative role of singlet oxygen in mediating UV-A induced generation of the photoagingassociated mitochondrial common deletion has been demonstrated in a pivotal study by Berneburg et al. [89]. Normal human fibroblasts were repetitively exposed to sublethal doses of UVA radiation and assayed for the common deletion. There was a time- and dose-dependent generation of the common deletion, attributable to the generation of singlet oxygen, since the common deletion was diminished when irradiating in the presence of singlet oxygen quenchers, but increased when enhancing singlet oxygen half-life by deuterium oxide. The induction of the
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common deletion by UVA irradiation was mimicked by treatment of unirradiated cells with singlet oxygen produced by the thermodecomposition of an endoperoxide. These studies provide direct evidence for the involvement of reactive oxygen species in the generation of agingassociated mtDNA lesions in human cells.
Side Effects of Anaerobic Energy Pathways High glycolytic fluxes and glucose accumulation may be a last resort when oxidative energy capacity of mitochondria declines during aging, but they are also sources of endogenous damage by themselves. In fact, both aging and diabetes are characterized by the formation of socalled advanced glycation endproducts (AGEs), though due to different reasons. Most glycolytic intermediates favour the formation of (AGEs) via reactive carbonyl groups, that are able to modify protein amino groups in the cytosol based on the Maillard reaction. This reaction is termed glycation or non-enzymatic glycosylation. AGEs constitute a heterogeneous group of structures, whereby N’-(carboxymethyl)lysine (CML) adducts are the most prevalent AGEs present in vivo. Methylglyoxal, glyoxal and other autooxidated derivatives of sugars induce AGEs that negatively affect essential features of skin cells and extracellular matrix proteins. It has been reported that AGE formation results in a loss of contractile capacity and cytoskeleton integrity in human skin fibroblasts, which possibly affects tissue cohesion and leads to visible effects of skin aging. Vimentin was identified as a major target in skin glycation besides other longlived proteins such as fibronectin, laminin, collagen, and elastin [90]. Strikingly, the accumulation of modified vimentin can be found in skin fibroblasts of elderly donors in vivo, bringing AGE modifications in skin into strong relationship with loss of organ contractile functions associated with aging. It is also reported that the intracellular concentration of the glycating agents – such as highly reactive methylglyoxyal, which is formed from dihydroxyacetone and glyceraldehyde-3-phosphates, and rapidly glycates proteins – damages mitochondria [91], and AGEs may even induce apoptosis by enhancement of expression of pro-apoptotic genes and stimulation of apoptosis through cytoplasmic and mitochondrial pathways [92]. Furthermore, intracellular AGEs induce oxidative stress, activate NF-kB and heme oxygenase, produce lipid peroxidation products, and cross-link proteins [93].
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Monitoring Methods for Energetic Processes in Skin Cells Monitoring of Mitochondrial and Glycolytic Activity in Skin There is no direct method available to measure mitochondrial or glycolytic activity in skin in situ. In any case, dermal cells have to be isolated from skin biopsies or suction blisters, or cultured dermal cell lines have to be employed. Assessment of the mitochondrial membrane potential is then possible through the availability of redistribution potentiometric radioactive compounds such as tetraphenylphosphonium bromide or redistribution potentiometric dyes such as rhodamine 123 (R123), or tetramethylrhodamine methyl ester (TMRM). These substances are lipophilic cations accumulated by mitochondria in proportion to the membrane potential (DC). Whereas the use of tetraphenylphosphonium bromide requires isolation of the mitochondria for uptale analysis [94], the dyes may be used also with living intact cells. Upon accumulation in the cytoplasm, R123 and TMRM exhibit a red shift in both their absorption and fluorescence emission spectra. The fluorescence intensity is quenched when the dyes are accumulated by mitochondria. These properties can be used to monitor membrane potentials even in single cells or in isolated mitochondria [95]. Rhodamine 123 and TMRM have also been used to monitor the mitochondrial membrane potential of intact human skin fibroblasts [96, 97], down to the single organelle level [98]. Another way to monitor the mitochondrial membrane potential is the use of redox indicators such as safranine, or the tetrazolium dye 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-benzimidazol-carbocyanine iodide (JC-1). The latter is taken up by cells and exists as a monomer in the cytosol (green). The negative charge established by the intact mitochondrial membrane potential allows the lipophilic dye, to enter the mitochondrial matrix where it accumulates. When the critical concentration is exceeded as the mitochondrial membrane becomes more polarized, J-aggregates form, which become fluorescent red and can be monitored photometrically. JC-1 has also been used to monitor the mitochondrial membrane potential on the level of single intact cells, for example by flow cytometry [99]. Another method to determine the energy flux in mitochondria is the estimation of the amount of ATP in relation to the amount of oxygen consumed, also called the P:O ratio. If mitochondria or permeabilized cells are incubated in an oxygraph apparatus (oxygen electrode) in
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an isotonic medium containing substrate and phosphate, then addition of ADP causes a sudden burst of oxygen uptake as the ADP is converted into ATP. The actively respiring state is refered to as State 3 respiration, while the slower rate after all the ADP has been phosphorylated to form ATP is refered to as State 4. The ratio [State 3 rate]: [State 4 rate] is called the respiratory control index and indicates the tightness of the coupling between respiration and phosphorylation. It is possible to calculate P:O ratio by measuring the decrease in oxygen concentration during the rapid burst of state 3 respiration after adding a known amount of ADP [16]. In parallel, the degree of glycolytic activity in a given cell suspension can be assessed by comparing the glucose uptake of cells with the amount of lactate produced.
Monitoring of Reactive Oxygen Species Although many enzymatic and chemical methods have been developed for evaluating ROS, in cell homogenates and in cell suspensions down to the single cell level, evaluation methods for ROS generation in situ are quite limited. Intracellular ROS production in single cells, for example, can be measured by a fluorometric assay based on deacetylation and oxidation of non-fluorescent dichlorodihydrofluoresceindiacetat (DCHF-DA), which penetrates the plasma membrane followed by enzymatic cleavage of the acetate groups, and which specifically reacts with peroxides to the fluorescent Dichlorofluorescein (DCF). Dihydrocalcein (H2-calcein) is another probe for intracellular ROS detection. In contrast to dichlorodihydrofluorescein, its fluorescent oxidation product calcein is thought not to leak out of cells. Other methods to monitor ROS in cells rely on luminescence enhancers such as luminol-, coelenterazine-, or lucigenin-enhanced chemiluminescence, which have been used extensively as indicators of O2● generation in intact cells and homogenates. Luminescence in this case is based on the activation of the probes by O2● and subsequent release of photons which can be measured in a luminometer. In situ, oxidative modification of dermal biomolecules induces ultraweak photon emission (UPE) as a by-product. Such light signals, which can be recorded in a non-invasive way, are supposed to contain valuable information regarding the extent of chemical damage, the nature of the oxidative modifications, and might be employed as a sensitive tool to monitor the efficacy of cosmetic or dermatological antioxidative intervention regimens. Data generated in indicate that UPE is induced by oxidative damage especially in deeper (living) skin layers, where antioxidants must be active in order to interfere with accelerated skin aging [100, 101].
Energetic Entry Points for Intervention There are several entry points for cosmetic intervention with intrinsic and extrinsic skin aging. Many of them refer to the topical application of antioxidants aimed at neutralizing free ROS, which are the central nocious effector molecules in both intrinsic and extrinsic aging processes. Typically used antioxidants such as Vitamin C, Vitamin E, Vitamin A and carotenes, have only indirect effects on energy metabolism, as they may reduce the ROS exposition of skin cells. These typical antioxidants will not be discussed in detail in this chapter because they are discussed extensively elsewhere in this book. There are, however, two compounds that may be topically used in cosmetic applications and dermatology, which interfere directly with the energy metabolism of skin cells – Creatine and Coenzyme Q10. Both compounds are in the very scope of this chapter and deserve in-depth discussion.
Energetic Effects of Creatine Creatine (Cr), a body-inherent amino acid derivative, is known to play a pivotal role in organ energy supply, because it acts like an energy store which can fastly provide energy in situations of high energy demand. After cellular uptake, creatine is phosphorylated to phosphocreatine (PCr) by the creatine kinase (CK) reaction using ATP. At subcellular sites with high energy requirements, e.g. at the myofibrillar apparatus during muscle contraction, CK catalyzes the transphosphorylation of PCr to ADP to regenerate ATP, thus preventing a depletion of ATP levels. PCr is thus available as a secondary energy source, serving not only as an energy buffer but also as an energy transport vehicle. In humans, the major part of the total creatine content is located in skeletal muscle, of which approximately a third is in its free form. The remainder is present in the phosphorylated form. It is supposed, for example, that the energy required for a 100-m sprint is entirely delivered from the creatine/phospho-creatine battery. Numerous scientific studies indicate that nutritional creatine supplementation favourably affects long-endurance exercise Vandenberghe et al. [102] and exerts protective effects in many clinical disorders, presumably caused by its energetic capacity. Creatine is normally metabolised to creatinine which is cleared via renal excretion, and daily turnover of creatine to creatinine for a 70-kg male has been estimated to be around 2 g. Cells attain their physiological levels of creatine, either by biosynthesis from the amino acids arginine, glycine, and methionine in the kidney, liver,
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and pancreas of vertebrates including humans, and/or alternatively by ingestion of meat and fish. Creatine is transported via the blood circulation and is taken up into cells by a Na- and Cl-dependent CRT protein [103, 32, 33]. Once inside a cell, creatine can be stored at high concentrations (e.g., 40 mM for muscle cells). Creatine can be synthesized in human cells, but from the age of about 30, a reduction in the cellular concentration in the skin can be determined [38]. Highly interesting with regard to aging processes is that mitochondrial creatine kinase activity prevents reactive oxygen species generation due to a kind of ‘‘antioxidant’’ role of mitochondrial kinase-dependent ADP re-cycling activity. Of course, creatine is not a radical scavenger in itself. Activation of the mitochondrial creatine kinase (mt-CK) by creatine and ATP or ADP rather induces a State 3-like respiration in mitochondria, which represents a condition where the rate of oxidative phosphorylation is not limited by ADP concentration [104]. Thus, supplementation of cells with creatine may reduce the electron ‘‘jam’’ in the respiratory chain by providing a sink for free ATP-coupled energy by building-up phosphocreatine (PCr) stores and consequently increasing the pool of available ADP for phosphorylation. Supplementation of skin cells with creatine has been described to reduce the amount of cellular damage induced by UV-A irradiation or chemical oxidants per se [17]. This protective effect is most likely due to the general energy-recharging effect of creatine and may have further implications in modulating processes which are involved in premature skin aging and skin damage. In contrast to the protective effect of creatine supplementation, inhibition of cytosolic and mitochondrial creatine kinase by siRNA in HaCaT- and HeLaS3-cells, as performed at the authors’ institution, affects cell viability and mitochondrial morphology negatively [105]. Besides its role on energy metabolism it has recently been demonstrated that activation of mt-CK by creatine inhibits the mitochondrial permeability transition (MPT), a process that is involved in apoptosis [106]. The postulated protective mechanism of mt-CK activity against MPT pore opening lies on the one hand on functional coupling between the mt-CK reaction and oxidative phosphorylation. It is known that MPT can be directly induced by mitochondrial ROS and it is conceivable that the protective role of mt-CK activity against MPT may occur through reduction of ROS generation by keeping ADP phosphorylation. On the other hand, octamer-dimer transitions of mt-CK as well as different creatine kinase substrates have a profound influence on controlling mitochondrial permeability transition (MPT). Kinetic
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analyses suggested a functional interaction between the mt-CK, outer membrane pore protein, and inner membrane adenylate translocator (ANT). Permeability transition-pore-like functions are not observed unless the creatine kinase octamer is dissociated, which is facilitated in the absence of creatine [107, 108, 109]. As a zwitter-ion, creatine is also able to penetrate skin remarkably well, and is able to replenish energy stores of epidermal cells. Several research data clearly demonstrate that only when enough creatine is available, skin cells function perfectly, all repair and protection systems work faultlessly, and the metabolism runs at full performance. For example, supplementation of keratinocytes in vitro with creatine has marked protective effects against oxidative stress, and in clinical trials conducted at the authors’ institution, exogenous supplementation of the skin with creatine in a topical formulation had marked protective against UV-induced damage [17]. In these studies, healthy old volunteers with an average age of 65.2 years were topically treated with a stabilized creatine formulation twice a day for 4 weeks on their upper arm. Afterwards, epidermal cells were isolated via suction blister to examine the mitochondrial membrane potential in response to UV irradiation. Epidermal cells from placebo-treated skin sites showed a substantial and statistically significant decline in their mitochondrial membrane potential compared with non-irradiated control cells. In contrast, cells from the creatine/creatinine-treated skin sites showed a statistically significant maintenance of their mitochondrial membrane potential even after irradiation compared with the irradiated placebo control cells. Thus, topically applied creatine significantly protects human epidermal cells from a UV-induced decline in mitochondrial energy metabolism. Moreover experimental results indicate that creatine promotes protection and repair of mitochondrial DNA to ensure the mainainance of healthy cells. As discussed in the section Genetic Damage to Mitochondria of this overwiew, mitochondrial mutations are thought to be mediated by ROS and persist in human skin as long-term biomarkers of UV exposure. In a pivotal study by Berneburg et al. [110], UV-induced mitochondrial mutagenesis of skin cells, as assessed by the frequency of the common deletion, as well as functional consequences on mitochondrial energy metabolism, could be normalized by increasing intracellular creatine levels. All these data clearly indicate that human skin cells energetically recharged with creatine both in vitro and in vivo are better protected against a variety of cellular stress conditions by reversing deficiencies in cutaneous energy supply [25]. So far there are no reports of harmful side effects of topical Cr loading of human skin.
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Energetic Effects of Coenzyme Q10 Coenzyme Q10 (CoQ10, ubiquinone) is a lipophilic vitamin-like substance which is present in most eukaryotic cells, primarily in the mitochondria [111]. The CoQ10 found in humans is a benzoquinone with a polyisoprene chain containing ten isoprene units. It is a component of the electron transport chain, where CoQ10 has a unique function since it transfers electrons from the primary substrates to the oxidase system at the same time that it transfers protons to the outside of the mitochondrial membrane, resulting in the proton gradient across the mitochondrial membrane which is employed by Comples IV to generate ATP. It is known that Complex I (NADH-ubiquinone reductase) and complex II (succinate-ubiquinone reductase) are found to be the predominant generators of ROS during prolonged respiration under uncoupled conditions, whereas complex III (ubiquinol:cytochrome c reductase) plays a less dominant role for mitochondrial ROS production. Complex II, in particular, appears to contribute most to the basal production of ROS in cells [112]. It is obvious that reduction of CoQ10 in a cell impairs the respiratory chain in mitochondria, resulting in electron ‘‘jam’’ and excess production of ROS. By reduction of the quinone to quinol, a carrier of protons and electrons is produced. Thus, in addition to its ‘‘antioxidative’’ role in the respiratory chain, the reduced form of CoQ10, ubichinol-10, is itself a real lipid-soluble radical scavenger molecule. It could be demonstrated in this context, that ubiquinone reduced to ubiquinol through the electron transport chain strongly inhibits lipid peroxidation in isolated mitochondria [113, 114]. Thus, reduced CoQ10 interferes also as an antioxidant with some of the basic age associated processes in mitochondria such as destruction on cardiolipin. Ubiquinol10 is about as effective in preventing peroxidative damage to lipids as Vitamin E, which is considered the best lipidsoluble antioxidant in humans. In contrast to Vitamin E, ubiquinol-10 is not recycled by ascorbate. However, it is known that ubiquinol-10 can be recycled by electron transport carriers present in various biomembranes and possibly by some enzymes [115]. In addition to direct antioxidant radical scavenging, the quinol can rescue tocopheryl radicals produced by reaction with lipid or oxygen radicals by direct reduction back to tocopherol (Vitamin E). Without CoQ10 in a membrane, regeneration of tocopherol is very slow. The regeneration of tocopherol can also be observed in low density lipoprotein
where a small amount of CoQ10 protects a larger amount of tocopherol [111]. In normal healthy individuals CoQ10 is synthesized in all cells from tyrosine (or phenylalanine) and mevalonate, and supplementation with CoQ10 does not increase tissue levels above normal. However, CoQ10 contents in cells decline during aging, which has been shown for example in muscle cells [116]. Previously it has beenshown that levels of CoQ10 are also lowered in skin cells from aging donors [117], suggesting that a decrease in mitochondrial CoQ10 content is an integral aspect of skin aging. In some tissue, such as aged skin, supplemental CoQ10 can restore normal levels. In fact, it was demonstrated that CoQ10 penetrates into the viable layers of the epidermis and reduces the basal level of oxidation measured by weak photon emission. Furthermore, a reduction in wrinkle depth following CoQ10 application was shown in clinical studies [117]. It is further known that UVirradiation depletes CoQ10 as well as other antioxidants in skin and causes oxidative damage [118]. In studies using nude mice, supplementation with CoQ10 was found to reduce the acute oxidative stress response following UV-irradiation, as characterized by reduced induction of manganese superoxide dismutase and glutathione peroxidase following irradiation in the presence of topical CoQ10 supplementation [119]. In studies by the authors, supplemental CoQ10 was also shown to be effective against UVA mediated oxidative stress in human keratinocytes in terms of thiol depletion, modulation of specific phosphotyrosine kinases and prevention of oxidative mtDNA damage [117]. CoQ10 was also able to significantly suppress the expression of collagenase in human dermal fibroblasts following UVA irradiation. In another study, conducted in the author’s institution, healthy volunteers were topically treated with a CoQ10-containing creme formulation twice a day in a 7-day period. Afterwards, epidermal primary keratinocytes were isolated via suction blister, irradiated and examined for mitochondrial membrane potential. CoQ10 application clearly resulted in a significant amelioration (+44%) of mitochondrial membrane potential after irradiation compared to the untreated control. These results indicate that CoQ10 has the efficacy to prevent many of the detrimental effects of photoaging and has general ‘‘energizing’’ effects in skin [117, 120].
Conclusion Cutaneous aging is characterized by a decline in energy metabolism of skin cells partially caused by detrimental
Alterations of Energy Metabolism in Cutaneous Aging
changes in mitochondrial respiration. The processes involved seem to be predominantly mediated by free radical actions known to be generated either by exogenous noxes such as UV light, or by endogenous processes such as impaired mitochondrial respiration associated with electron ‘‘jam’’ and generation of ROS by leakage of electrons from the respiratory chain. It is widely accepted that alterations in mitochondrial respiration can be regarded as both a reason as well as an important consequence for aging. Any lack of mitochondrial function impairs cellular ATP synthesis, reducing the ‘‘fuel supply’’ for repair mechanisms. It does further induce the formation of ROS as byproducts of an impaired mitochondrial respiration. Accumulation of ROS may, in turn, damage neighbouring mitochondrial complexes, membranes and mtDNA and further accelerate the aging process in a kind of feedback loop. Once the damage of macromolecules has reached the level of mtDNA, leading to mutations, the energetic age of a mitochondrium, and thus of a cell, is carved in stone. Basically, any loss of mitochondrial energetic capacity is attempted to be compensated by energy generation from other sources. This is either the exploitation of intracellular energy stores for high energy demands in a short term, or the swith to anaerobic pathways for energy supply, such as glycolysis, as a last resort in a long term. In this context glycolysis, used by a cell as last resort, is associated with the generation of reactive glycolytic intermediates which favour the formation of advanced glycation endproducts (AGEs) via reactive carbonyl groups. These AGEs may harm a cell by processes ranging from the generation of infunctional cytoskeletal proteins up to the induction of apoptosis. Thus, it is important to keep tissues from anaerobiosis by keeping mitochondrial energy generation upright. Furthermore, it is important to supply a cell with substantial energy stores to be filled with energy in phases of baseline activity, which can be used in situation of high energy demand without the need to swith to glycolysis. There are several entry points to affect skin aging from an energetic perspective. Besides sunscreens to protect the skin from the most important ROS-generating insult, UV light (> Fig. 29.1A), the first and most obvious entry point is to keep the antioxidant system of a cell working. This will result in protection against ROS from whatever sources and thus targets the key effector molecules of skin aging (> Fig. 29.1B). Antioxidants are beyond the scope of this overview. However, it should be mentioned that one antioxidant which has been proven to be especially potent in pathological dermatological situations characterized by excessive deregulation of ROS generation, such as
29
polymorphous light eruption (PLE), is a-glucosylrutin [121, 122, 123], a potent plant-derived antioxidant with excellent bioavailability in skin. The next energetically relevant entry point with regard to skin aging is to keep the respiratory chain in skin cells working, in order to avoid electron ‘‘jam’’ and subsequent generation of ROS. This can be done by supplementation with Coenzyme Q10, which is a key component of the respiratory chain. CoQ10 declines with age but can be effectively replenished in skin by topical formulations (> Fig. 29.1C). In addition to its central role as a structural component of the respiratory chain, CoQ10 is also a potent radical scavenger which protects important membrane proteins of the respiratory chain, such as cardiolipin, from oxidative damage. In addition to this, supplementation of skin with creatine is an important entry point for intervention with aging processes. Creatine levels and activities of creatine kinases in skin decline with age, reducing the available energy store for short term demand. Skin cells that are energetically re-charged with sufficient creatine are better protected against a variety of cellular stressors, age-dependent deficiencies in cellular functions, or oxidative and free-radical-induced cell damage. This is also due to a kind of ‘‘antioxidant’’ activity of the creatine/phosphocreatine system, which results from its capacity to form a sink for ATP, keeping the amount of available ADP for phosphorylation by the mitochondrial respiratory chain high (> Fig. 29.1D). This, in turn, keeps the mitochondrium in a kind of unrestricted State 3 respiration. At first sight, it might be tempting to speculate that supplementation of skin with additional oxygen might also enforce the capacity of the respiratory chain to generate energy. This, however could not yet be proven in experimental systems or clinical trials, and it might be due to the fact that skin can directly take up atmospheric oxygen in sufficient amounts, thus is generally not in a situation of hypoxia as other organs might be [20]. Nevertheless, beneficial effects of short term oxygen pulses on the activity of skin cells have been observed, but these are supposed to be related to some yet unknown signal transduction effects of oxygen, rather than on enhanced energy metabolism (publication in preparation). The energetic pathways leading to skin aging, as well as the possible intervention strategies, as discussed in this overview, are summarized in > Fig. 29.1. In conclusion, biological science has advanced the ability to directly target skin aging. The declining energy
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. Figure 29.1 Pathways leading to aging of skin cells. Processes (A)–(D) are described in detail in the Conclusion of this overview. Blue arrows marked with a (+) sign represent beneficial processes for the cell with anti-aging efficacy. Red arrows marked with a () sign represent detrimental processes for a cell, which are associated with accelerated aging. ROS = reactive oxygen species; AGE = advanced glycation endproduct; RC = respiratory chain; CoQ10 = Coenzyme Q10; mtDNA = mitochondrial DNA; Cr = creatine; PCr = phosphocreatine
Alterations of Energy Metabolism in Cutaneous Aging
29
. Figure 29.1 (Continued)
metabolism has turned out as a high priority field of anti-aging interventions such as creatine and CoQ10.
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115. Frei B, Kim MC, Ames BN. Ubiquinol-10 is an effective lipidsoluble antioxidant at physiological concentrations. Proc Natl Acad Sci USA. 1990;87:4879–4883. 116. Lass A, Kwong L, Sohal RS. Mitochondrial coenzyme Q content and aging. Biofactors. 1999;9:199–205. 117. Hoppe U, Bergemann J, Diembeck W, et al. Coenzyme Q10, a cutaneous antioxidant and energizer. Biofactors. 1999;9:371–378. 118. Podda M, Traber MG, Weber C, Yan LJ, Packer L. UV-irradiation depletes antioxidants and causes oxidative damage in a model of human skin. Free Radic Biol Med.1998;24:55–65. 119. Kim DW, Hwang IK, Yoo KY, et al. Coenzyme Q_{10} effects on manganese superoxide dismutase and glutathione peroxidase in the hairless mouse skin induced by ultraviolet B irradiation. Biofactors. 2007;30:139–147. 120. Stab F, Wolber R, Blatt T, Keyhani R, Sauermann G. Topically applied antioxidants in skin protection. Methods Enzymol. 2000;319:465–478. 121. Hadshiew IM, Treder-Conrad C, v Bulow R, et al. Polymorphous light eruption (PLE) and a new potent antioxidant and UVAprotective formulation as prophylaxis. Photodermatol Photoimmunol Photomed. 2004;20:200–204. 122. Rippke F, Wendt G, Bohnsack K, et al. Results of photoprovocation and field studies on the efficacy of a novel topically applied antioxidant in polymorphous light eruption. J Dermatolog Treat. 2001;12:3–8. 123. Wolber R, Stab F, Max H, et al. Alpha-glucosylrutin, a highly effective flavonoid for protection against oxidative stress. J Dtsch Dermatol Ges. 2004;2:580–587.
3 Basophilic (Actinic) Degeneration of the Dermis: An Easy Histological Scoring Approach in Dermal Photoaging Peter Helmbold
Introduction Chronic ultraviolet (UV) light exposure of skin leads to typical effects: changes in the collagen and elastic tissue matrix is considered the characteristic histological finding in aged skin, followed by visible wrinkling and pigmentary changes. Changes in the epidermis include thinning to atrophy, hyperplasia of melanocytes, and disturbances in the texture of keratinocytes. Assessment of the degrees of photoaging by a grading system with low interobserver coefficient of variation seems to be of special interest. Different clinical methods have been proposed including descriptive grading clinical scales, visual analogue scales, and photographic grading scales [1]. Some of these methods like ‘‘skin surface topography grading’’ [2] were compared with histological changes like actinic elastosis. Other studies used histological scoring of dermal aging independent of a noninvasive scoring system. The following approaches were used: quantification of elastic tissue [3], type III procollagen, type III to type I procollagen ratio, quantification of the grenz zone (a wide band of eosinophilic material just beneath the epidermis, devoid of oxytalan fibers) [4], activated fibroblasts with positive procollagen staining [5], acid mucopolysaccharides, improved quality of elastic fibers, and increased density of collagen [6], quantification of changes in the epidermis (thinning of the stratum corneum, granular layer enhancement, and epidermal thickening) [7]. One disadvantage of most of these methods is that actinic and instrinsic aging cannot be distinguished from one another. Bhawan et al. [8] systematically investigated histological effects of photoaging. The following features proved to be significantly changed in photoaged skin: increase in melanocytes, increase in melanocytic atypia and epidermal melanin, reduced epidermal thickness, more compact stratum corneum, increased granular layer thickness,
increased solar elastosis, dermal elastic tissue, melanophages, perivascular inflammation, and perifollicular fibrosis but no change in the number of mast cells or dermal mucin in the photoexposed skin. Of these, actinic elastosis (basophilic degeneration of the dermis) was the single most reliable factor. Basophilic degeneration is very consistent with the clinical sign of wrinkling and with dermal microvasular aging (see Chapter 2). Thus, a single-factor scoring system of dermal aging regarding dermal basophilic degeneration (DBD) was developed. It should be mentioned that the knowledge of dermal fiber degeneration is not new and the use of a scoring system is the result of previous work [9, 10]. After first experiments with a five-level system, it was found that best interobserver agreement was obtainable with a three-level model (> Table 3.1) together with a histological atlas of the different levels (> Fig. 3.1). This model was tested in 120 biopsies from normal skin of 87 patients (42 females, 45 males, 27.9 23.7 years [mean SD]) from surplus areas (i.e., Burow’s triangle) of routinely excised and histologically controlled benign nevus cell nevi of normal skin. Each specimen was
. Table 3.1 Histological scoring of dermal basophilic degeneration (DBD) [11] No actinic damage (Level 0): No fiber degeneration (> Fig. 3.1a, b). Moderate actinic damage (Level 1): Fragmentation of fibers of the upper dermis and presence of single basophilic fibers (> Fig. 3.1c, d). High actinic damage (Level 2): Spotted or band-like basophilic degeneration with conglomerates of basophilic masses in the upper and/or mid-dermis (> Fig. 3.1e, f).
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. Figure 3.1 Scoring of dermal basophilic degeneration (DBD). (a, b) No fiber degeneration (DBD level = 0). (c, d) Fragmentation of fibers (arrows) of the upper dermis and presence of single basophilic fibers (DBD level = 1). (e, f) Spotted (e) or band-like (f) basophilic degeneration (arrows) with conglomerates of basophilic masses in the upper and mid-dermis (DBD level = 2). Scale bar: a, c–f = 200 mm, b = 100 mm (Helmbold et al. [11]. Reprinted with permission of J Invest Dermatol)
characterized by a set of clinical data: age, sex of the patient, and body location of the biopsy with regard to typically solar-exposed skin areas. The interobserver reliability (agreement among four independent observers) of this technique was 92.2 4.6%
in all biopsies. There was no disagreement of more than one level between the investigators. Correlations were found between DBD and the age of the patient (Spearman r = 0.662, p < 0.001) as well as DBD and body regions with typical chronic solar exposure (Spearman r = 0.244,
Basophilic (Actinic) Degeneration of the Dermis: An Easy Histological Scoring Approach in Dermal Photoaging
p = 0.005). Sixty-eight biopsies revealed no visible DBD (37 from female, 31 from male patients; age: 19.8 18.4 years), 36 biopsies showed moderate ‘‘level 1’’ DBD (28 females, 10 males, 39.9 19 years), and 16 had a high ‘‘level 2’’ DBD (six females, ten males, 64.4 11.9 years). DBD was not observable in patients younger than 15 years.
Conclusion The advantages of this approach are easy application, use of HE-stained routine sections, fast determination, and sure results with high interobserver agreement. Disadvantages are that this approach cannot ‘‘measure’’ minimal differences and that it reflects only the dermal component of photoaging. Thus, the mean application fields are the studies that need reliable classification if there is actinic degeneration (or not). On the other hand, this approach is not suitable for quantification of the effects of an anti-aging product or similar studies.
Cross-references > Histology
of Microvascular Aging of Human Skin
References 1. Kappes UP. Skin ageing and wrinkles: clinical and photographic scoring. J Cosmet Dermatol. 2004;3:23–25.
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2. Battistutta D, Pandeya N, Strutton GM, et al. Skin surface topography grading is a valid measure of skin photoaging. Photodermatol Photoimmunol Photomed. 2006;22:39–45. 3. Chiu AE, Chan JL, Kern DG, et al. Double-blinded, placebocontrolled trial of green tea extracts in the clinical and histologic appearance of photoaging skin. Dermatol Surg. 2005;31:855–860; discussion 860. 4. Seite S, Bredoux C, Compan D, et al. Histological evaluation of a topically applied retinol-vitamin C combination. Skin Pharmacol Physiol. 2005;18:81–87. 5. Rostan E, Bowes LE, Iyer S, et al. A double-blind, side-by-side comparison study of low fluence long pulse dye laser to coolant treatment for wrinkling of the cheeks. J Cosmet Laser Ther. 2001;3:129–136. 6. Ditre CM, Griffin TD, Murphy GF, et al. Effects of alpha-hydroxy acids on photoaged skin: a pilot clinical, histologic, and ultrastructural study. J Am Acad Dermatol. 1996;34:187–195. 7. Newman N, Newman A, Moy LS, et al. Clinical improvement of photoaged skin with 50% glycolic acid. A double-blind vehiclecontrolled study. Dermatol Surg. 1996;22:455–460. 8. Bhawan J, Andersen W, Lee J, et al. Photoaging versus intrinsic aging: a morphologic assessment of facial skin. J Cutan Pathol. 1995;22: 154–159. 9. Suwabe H, Serizawa A, Kajiwara H, et al. Degenerative processes of elastic fibers in sun-protected and sun-exposed skin: immunoelectron microscopic observation of elastin, fibrillin-1, amyloid P component, lysozyme and alpha1-antitrypsin. Pathol Int. 1999;49: 391–402. 10. Lund HZ, Sommerville RL. Basophilic degeneration of the cutis; data substantiating its relation to prolonged solar exposure. Am J Clin Pathol. 1957;27:183–190. 11. Helmbold P, Lautenschlager C, Marsch W, et al. Detection of a physiological juvenile phase and the central role of pericytes in human dermal microvascular aging. J Invest Dermatol. 2006;126: 1419–1421.
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35 Biological Effects of Estrogen on Skin Zack Thompson . Howard I. Maibach
Introduction Changes in skin aging and function occur at variable rates, and are influenced by environmental, hormonal, and genetic factors unique to each individual. Skin aging involves progressive degenerative changes, such as gradual dryness, thinning, fragility, atrophy, and wrinkling. Over time, the skin experiences a progressive increase in extensibility and a reduction in elasticity, thereby becoming more frail and susceptible to trauma. This in turn leads to an increased risk of skin injury (e.g., lacerations, tears, ulcerations, bruising), and an impairment of wound healing. Improvements in nutrition, sanitation, quality and provision of healthcare, and other related factors have led to a dramatic increase in life expectancy for human beings over the past century. The average life expectancy of women in the United States has increased from roughly 50.7 years in 1900 [1], to an estimated 80.7 years by 2009 [2]. As the developing world catches up, a similar but much more significant trend in average global longevity is projected to occur. The number of women age 60 and over worldwide is expected to increase from about 336 million in 2000, to just over 1 billion by 2050 [3]. Since the average woman in a developed nation spends about one third of her life after the onset of menopause, the benefits and risks of estrogen replacement therapy (ERT) – also known as hormone replacement therapy (HRT), and menopausal hormone therapy (MHT) – have become major areas of focus for research. ERT has been regularly prescribed by physicians for postmenopausal women since the 1940s to reduce symptoms associated with menopause, such as hot flashes, night sweats, vaginal dryness, and sleep disturbances [4]. In the intervening period, the risks and benefits of ERT have been and continue to be debated. In terms of benefits to ERT, estrogen has been demonstrated to ameliorate menopausal maladies such as: vasomotor symptoms, mood changes, atrophy of reproductive organs, and sleep disturbance [5]. More recently, the Women’s Health Initiative (WHI) studies were conducted to assess the risks and benefits from ERT. However, the WHI did not perform any
analysis of the effect of ERT on skin in terms of cosmetic appearance, morbidity, or mortality. Additionally, the WHI studies examined only oral conjugated equine estrogens (CEE) 0.625 mg and medroxyprogesterone acetate 2.5 mg, not topical or transdermal ERTs, which could have a more focused impact on the skin. Particularly in light of the increasing life expectancy of women throughout the world, health workers need to more fully understand the physiology and treatment of menopause. To this end, this chapter focuses on the biological effects of estrogen on the skin of postmenopausal women with regard to skin thickness, moisture, wrinkling, wound healing, and scarring, and briefly discusses future estrogen therapies, such as selective estrogen receptor modulators (SERMs). Studies have uncovered various mechanisms by which estrogen may affect skin aging and function. Research indicates that topical and systemic ERT lead to a statistically significant improvement in many aging skin problems [6]. ERT increases skin collagen content and preserves thickness, thereby reducing wrinkling. Skin moisture content improves with ERT, as it increases the skin’s hyaluronic acid, acid mucopolysaccharides, and sebum levels, and possibly maintains stratum corneum barrier function. Beyond its impact on aging, topical ERT accelerates and improves cutaneous wound healing in elderly individuals, possibly by regulating the levels of a cytokine. Conversely, a lack of estrogen (i.e., hypoestrogenism) or addition of tamoxifen – the first SERM developed – may improve the quality of scarring, though the relationship between estrogen and scarring is more ambiguous.
Thickness and Collagen Collagen is the primary protein of connective tissue in mammals, comprising about 25–35% of total body protein content. Collagen has a high tensile strength, and is a major component of fascia, cartilage, ligaments, tendons, bone, and skin. At least 30 different collagen genes have been identified and described. These collagen genes combine to form over 20 different types of collagen fibrils, of
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which Types I, II, and III are the most common [7]. Human skin contains over 14 types of collagen, of which 80% is Type I – the collagen that is responsible for skin strength – and 15% is Type III – the collagen that is responsible for the elastic properties of skin. Collagen becomes progressively sparse, disordered, and atrophied as skin ages – one of the chief reasons for the skin transformations resulting from aging. Copious studies establish menopause leads to estrogen deficiency, and research over the past 60 years demonstrates skin
thickness, estrogen content, and skin collagen are closely correlated (> Table 35.1). In 1941, Albright et al. first noticed elderly women with osteoporotic fractures – an injury closely correlated with menopause – had visibly thinner skin. Correlation between skin thickness and estrogen content was first noted by Bullough’s 1941 study on mice [8]. McConkey et al. posited in 1963 that the ‘‘transparent skin’’ described by Albright was caused by a decrease in dermal collagen Type I [9]. In 1970, Black et al. [10] demonstrated skin
. Table 35.1 Selected studies on skin thickness and ERT Date
Study
1969 Rauramo and Punnomen
Type of measurement
Hormones used
Skin biopsy analysis Estradiol succinate (measured by calipers) 2.0 mg
Duration
Results
6 months
Improvement in skin thickness
1987 Brincat et al. Skin biopsy analysis
Estradiol implant and percutaneous estradiol gel
2–10 years
Increase in skin thickness by 30%; average linear decline of 1.13% skin thickness and 2.1% collagen per year in the first 15–18 postmenopausal years without ERT
1992 CasteloSkin biopsy analysis Branco et al.
Conjugated equine estrogens or transdermal 17bestradiol
12 months
Increase in skin collagen by 1.8–5.1%
1994 Maheux et al.
Skin thickness (measured by ultrasonagraphy)
Conjugated estrogen 0.625 mg
12 months
Increase in skin thickness by 11.5%
1995 Varila et al.
Skin biopsy analysis
Topical 17b-estradiol
3 months
Increase in hydroxyproline by 38%
1996 Callens et al. Skin thickness 17b-estradiol gel or (measured by estradiol patches ultrasonagraphy at five skin locations)
Mean of Increase in skin thickness by 7–15% in all 4.8 years locations
2000 Sauerbronn et al.
Skin biopsy analysis
2.0 mg valerate estradiol, cycled with 1.0 mg cyproterone acetate
6 months
Increase in skin collagen by 6.49%
2007 Sator et al.
Sebumeter, the Corneometer, and high-frequency ultrasound
2.0 mg 17b-estradiol/ 10 dydrogesterone
7 months
Significant improvements in skin elasticity, skin hydration, and skin thickness
1993 Sawwas and Skin biopsy analysis Laurent
Subcutaneous estradiol and testosterone
3–14 years
Significantly greater levels of collagen Type III
1997 Haapasaari et al.
17b-estradiol and norethisterone acetate; estradiol valerate
12 months
No significant change in skin collagen or thickness
Skin biopsy analysis
Biological Effects of Estrogen on Skin
thickness is in fact proportional to collagen content – as McConkey et al. had suggested – utilizing the 1964 radiographic technique for measuring skin thickness employed by Meema et al. [11]. The above studies established that the estrogen deficiency associated with menopause leads to skin collagen degradation and decreasing skin thickness. Thus, future studies focused on deciphering the effects of reproductive hormones on skin collagen. In 1969, Rauramo and Punnomen noted ERT had a favorable effect on human skin [12]. Their study involved 6 months of treatment with 2.0 mg estradiol succinate, and showed improvements in skin thickness of biopsies measured using calipers. A subsequent 1987 study by Brincat et al. corroborated Rauramo and Punnomen’s findings, showing a decrease in skin thickness and collagen content after menopause. Their study demonstrated an increase in skin thickness for women receiving an estradiol implant or percutaneous estradiol gel. Using radiographic techniques, Brincat et al. found an average increase in skin thickness by 30% for those on ERT. Additionally, Brincat et al. found 30% of collagen is lost within the first 5 years of menopause, and witnessed an average linear decline of 1.13% in skin thickness and 2.1% in collagen per year in the first 15–18 postmenopausal years for those not on ERT. The study also noted that skin collagen decline was correlated specifically to the duration of estrogen deficiency (i.e., postmenopausal years), and not chronological age [13]. In a 1992 study, Castelo-Branco et al. similarly found that ERT – both oral and transdermal – increased skin collagen content. Their study used conjugated equine estrogens and transdermal 17b-estradiol over a 12 month period, and showed an increase in skin collagen content of 1.8–5.1%, varying based on the type of ERT administered. Unlike Brincat et al., Castelo-Branco et al. observed a higher correlation between skin collagen content and chronological age, though they still recognized a statistically significant correlation between skin collagen content and time since the onset of menopause [14]. Subsequent studies by Affinito et al. and others have achieved the same results as Brincat et al., and showed postmenopausal years to be the determining factor [15]. The likely cause of this aberration in the Castelo-Branco et al. study was that many participants were in their initial postmenopausal years, with a short history of estrogen deprivation. This problem with study participants has plagued other studies (e.g., [16, 17]), and has contributed to some of the controversy surrounding the effects of ERT on skin aging. A 1994 study by Maheux et al. controlled for some of the factors which complicate the measurement of skin aging
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(e.g., smoking, exposure to solar radiation), thereby focusing more specifically on the effects of conjugated estrogens. Maheux et al. employed a randomized, double-blind, placebo-controlled study of postmenopausal nuns. Their study used conjugated estrogen 0.625 mg over a 12 month period, and showed an increase in skin thickness, as measured by ultrasonography, of 11.5% for the group receiving ERT [18]. Varila et al. studied the effect of topical ERT on collagen content, as measured by skin hydroxyproline, in a 1995 study. Hydroxyproline is a major component of the protein collagen, and is found in few proteins other than collagen. The only other mammalian protein that includes hydroxyproline is elastin. For this reason, hydroxyproline content has been used as an indicator to determine collagen or gelatin amount. Via skin biopsy analysis, Varila et al. measured an increase in hydroxyproline of 38% following administration of topical 17b-estradiol for 3 months. The study also observed increased levels of the carboxyterminal propeptide of human type I procollagen and of the aminoterminal propeptide of human type III procollagen, thus showing that estrogen increases collagen synthesis [19]. Subsequent studies by Callens et al. [20], Sauerbronn et al. [21], Sator [22] and others have further substantiated the aforementioned studies’ claims. Callens et al. found ERT increased skin thickness by 7–15% in postmenopausal women utilizing estradiol gel patches or an estradiol transdermal system. Sauerbronn et al. focused on skin collagen rather than thickness. Following 6 months of treatment with estradiol valerate and cyproterone acetate, their study observed a 6.49% increase in collagen fibers in the dermis, and no significant change in epidermal thickness. Sator noted significant improvements in skin elasticity, skin hydration, and skin thickness after 7 months of treatment. This corroborates a 2005 Sumino et al. study, which showed after menopause, skin elasticity declined 0.55% per year, and that 12 months of ERT increased elasticity by 5.2% [23]. While ERT increases skin thickness, there are limits to the potential for ERT to reverse skin aging. Savvas and Laurent conducted research in 1993 which, in accordance with the 1987 Brincat et al. study, suggests ERT produces no additional increase in skin collagen content after 2 years. Their study focused specifically on collagen Type I and Type III, and found increased collagen content in postmenopausal women receiving estradiol and testosterone implants. However, beyond 3 years they found no increase in the proportion of collagen Type III [24]. At least one notable study from 1997 appears to dispute the evidence that ERT increases collagen content and
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skin thickness. Utilizing immunohistochemistry and colorimetric methods, Haapasaari et al. detected no increase in skin collagen content following 1 year of systemic estrogen therapy. The investigators hypothesized estrogen affects collagen turnover rather than skin collagen [18]. A more likely explanation, however, is that the low median postmenopausal age of 12 months amongst the study participants did not provide adequate time to show the effects of low estrogen levels. Lastly, as seen earlier by the wide range of improvement, it is difficult to quantify the real effect of ERTs on skin thickness and collagen content. Some factors such as individual skin history are extremely difficult to correct for when studied. For example, Lee et al. highlighted the need to standardize the method of measuring skin atrophy and thickness so that results are more comparable across studies [25].
Moisture Studies show the loss of moisture, which leads to dry skin, is another age-related skin condition. In 1996, Schmidt et al. noted an increase in skin moisture amongst perimenopausal and postmenopausal women after using topical estradiol 0.01% and estriol 0.3% (systemic) for 6 months [26]. Dunn et al. compiled results from a much larger population based cohort study in 1997 – the first National Health and Nutrition Examination Survey (NHNES), a Centers for Disease Control and Prevention (CDC) program of studies designed to assess the health and nutritional status of adults and children in the United States – and showed postmenopausal women on ERT were significantly less likely to experience dry skin [27]. Research provides the following possible explanations as to why topical and systemic estrogen therapies preserve skin moisture: increased acid mucopolysaccharides and hyaluronic acid in the dermis, higher sebum levels, increased water-holding capacity of the stratum corneum, and changes in the corneocyte surface area. First, Grosman et al. observed increased acid mucopolysaccharides and hyaluronic acid in the skin of mice treated with estrogen in the early 1970s. Hyaluronic acid is known to have a high water holding capacity, which supports an increase in dermal water content [28, 29]. Second, the 1996 study by Callens et al. demonstrated a 35% increase in sebum levels amongst women on estrogen [19]. This study suggests that ERT may prevent the decrease in glandular secretions Pochi et al. noted in postmenopausal women in their 1979 study [30].
Third, the ability of skin to retain water is largely associated with the stratum corneum lipids, which maintain skin barrier function. In 1995, Pierard-Franchimont et al. suggested estrogen may play a role in stratum corneum barrier function, noting women on transdermal ERT showed increased water-holding capacity of the stratum corneum [31]. Similarly, Paquet et al. demonstrated in 1998 that estrogen also improves the ability of the stratum corneum to prevent water loss, by observing a decrease in the rate of water accumulation in postmenopausal women [32]. Lastly, ERT may lead to changes in the corneocyte surface area, thereby further enhancing the epidermal barrier function.
Wrinkling Skin wrinkling is the result of lost skin elasticity [33], dermal thickening, and elastic deterioration caused by a variety of degenerative environmental, hormonal, and genetic factors. Histological studies of wrinkles by ContetAudonneau et al. [34] and Bosset et al. [35] showed alterations of dermal collagen and elastic fibers, as well as a marked decrease in glycosaminoglycans. Numerous studies have shown ERT can improve fine wrinkles, prevent development of skin wrinkles, and decrease existing wrinkle depth. Creidi et al. studied the effect of conjugated estrogen cream on postmenopausal women in a double-blind, placebo-controlled study utilizing clinical evaluation by dermatologists. This study found significant improvement in fine wrinkles [36]. Dunn et al. employed the NHNES to control for age, body mass index, and sun exposure, and found postmenopausal women on ERT were less likely to develop wrinkles in the first place [26]. Schmidt et al. further demonstrated that existing wrinkle depth can be decreased using topical ERT [25]. In contradiction to these studies, a 2008 study by Phillips et al. found no improvement in age-related skin changes from topical ERT. However, this study was limited in that it utilized low-dose estrogen (i.e., 1 mg norethindrone acetate and 5–10 mg ethinyl estradiol) for only 48 weeks, and in women with an average of only 5 postmenopausal years [17]. The impact of ERT on wrinkling likely relates partially to its impact on collagen; namely its capacity to increase the proportion of collagen III in skin. Punnonen et al. noted the elastic fibers in the papillary dermis following local estriol treatment had thickened, increased slightly in number, and become better oriented [37].
Biological Effects of Estrogen on Skin
Furthermore, the increase in hyaluronic acid noted by Grosman increases water capacity and thus skin turgor, thereby reducing the ability to develop wrinkles and the appearance of any wrinkles present [28]. An interesting side note, however, is that a study by Castelo-Branco et al. found ERT does not appear to reduce wrinkling in those with a history of over 10 years smoking. Thus, it seems ERT cannot reverse some of the damage done by smoking, such as destruction of the ground substance, decreased blood flow in the skin, and direct toxic effects [38].
Wound Healing Natural aging significantly impacts wound healing by increasing susceptibility to trauma, bruising, and chronic wounds. The role of estrogen and effect of ERT on wound healing is only recently becoming understood, largely because most early studies were performed using animal models that produced conflicting results. Differences in species, duration of treatment, and methodologies employed likely led to the inconsistent findings in animal studies conducted during the 1960s and 1970s [39]. Recent studies on wound healing have focused primarily on the molecular role of estrogen on the cells and metabolic processes involved in wound repair. Age-related impairment of wound healing has been partially attributed to low levels of transforming growth factor-b1 (TGFb1), decreased collagen synthesis, and increased presence of proteases (specifically elastase). Furthermore, estrogen’s presence on fibroblasts – the main cell type involved in wound healing – indicates it may directly modify their function [39]. Studies by Ashcroft et al. suggest estrogen positively affects wound repair by causing TGF-b1 secretion by fibroblasts – not by increasing fibroblast production in a wound – increasing collagen content, and reducing collagenolysis [40–42]. The 1997 Ashcroft et al. study used rats to demonstrate topical ERT was associated with significantly accelerated acute wound healing as shown by decreased re-epithelialization time, decreased wound width, and increased collagen deposition. The 1999 and 2003 Ashcroft et al. studies demonstrated topical ERT reduces the activity of protease elastase in cutaneous wounds in humans, thereby improving healing as shown by decreased wound sizes, faster increases in collagen levels, increased fibronectin levels, and enhanced strength. Furthermore, the latter study suggested topical ERT may even be useful on a prophylactic basis, though more work needs to be done in this area.
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Scarring The cellular and subcellular sites and mechanisms involved in the scarring process are poorly understood. Whitby et al. conducted a study comparing the differences between fetal and adult wound healing. Fetal scars tend to be superior in that they are pale and flat, rather than pigmented and everted as in adults. Their study found fetal wounds deficient in the inflammatory cytokine TGFb1, whereas adult skin typically had a large amount of TGF-b1 [43]. As mentioned in the section on wound healing, postmenopausal women in a hypoestrogenic state tend to be deficient in TGF-b1; thus, they should produce scars that are better both macroscopically and microscopically. A study by Shah et al. supports this supposition, and suggests estrogen antagonists may be effective in limiting scarring [44].
Selective Estrogen Receptor Modulators (SERMs) SERMs act at the level of the estrogen receptors, either mimicking positive estrogen effects or blocking negative estrogen effects, depending on the tissue. This tissue specificity allows for targeted ERT treatments. For example, tamoxifen has an antiestrogenic effect on breast tissue but an estrogenic effect on bone, and is used to treat breast cancer and prevent postmenopausal osteoporosis simultaneously. Hu et al. demonstrated tamoxifen also inhibits collagen wound contraction, and indicated it affected fibroblast morphology. A potential mechanism for the inhibition of wound contraction could be the inhibition of fibroblasts or fibroblast proliferation [45]. Surazynski et al. found that in fibroblasts, a SERM currently used for the treatment of postmenopausal osteoporosis known as raloxifene has a stronger positive stimulating effect on collagen synthesis than estradiol [46]. Intensive research is currently underway to develop new SERMs specifically for the purpose of targeting the skin without incurring systemic side effects [47]. Thus, these drugs will likely become an important means of controlling scarring and other effects of skin aging.
Conclusion New studies continue to support the above conclusions that both topical and systemic ERT have positive impacts
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on skin aging as it relates to thickness, moisture, wrinkling, wound healing, and scarring [48–50]. ERT’s ability to slow skin aging is largely due to estrogen’s ability to repair and prevent a decline in skin collagen, increase skin turgor and water holding capacity, improve epidermal barrier function, and decrease changes in skin elasticity over time. Other areas remain to be explored further, such as optimal methods for administering ERT, determining the impact of ERT on men and premenopausal women, understanding the effect of estrogen on melanocyte function, and ascertaining the role of estrogen in skin cancer prevention, if any. ERT also affects skin and skin appendages in ways not discussed in this chapter, such as hair growth during pregnancy, and hair loss during menopause [51–53]. When determining if ERT is appropriate, the benefits and risks of ERT must be weighed by patients and healthcare workers in light of the potential side effects, particularly from systemic ERT. At this point in time, treatment of skin aging should not be the sole basis for systemic ERT, at least until it is understood what minimum concentration of estrogen can achieve the best local effects without systemic hormonal side effects. Most topical ERTs (e.g., estradiol creams) may not be significantly systemically absorbed [54]. Since topical ERT may present a safe and effective treatment for skin aging, menopausal women not receiving systemic ERT are candidates for this treatment when it is administered by a dermatologist educated in endocrinology. Finally, SERMs may present another future means of achieving the benefits of estrogen therapy without the systemic risks. Taken together, current experimental data suggests a careful prospective examination of topical estrogens to ameliorate skin aging. Today’s experimental tools should provide reliable efficacy and toxicity metrics.
Cross-references > Aging
Genital Skin and Hormone Replacement Therapy Benefits
References 1. World Health Organization Fact Sheet Number 252. http://www. who.int/mediacentre/factsheets/fs252/en/. June 2000. 2. Censtral Intelligence Agency. The World Fact Book. https://www.cia. gov/library/publications/the-world-factbook/geos/US.html. Updated Apr 30, 2009. 3. Women, Ageing and Health: A Framework for Action. World Health Organization. http://www.who.int/ageing/publications/ Women-ageing-health-lowres.pdf. 2007. 4. Shah M, Maibach H. Estrogen and skin, an overview. Am J Clin Dermatol. 2001;2(3):143.
5. Palacios S. Current perspectives on the benefits of HRT in menopausal women. Maturitas. 1999;33:S1–S3. 6. HRT/ERT refers to supplements of hormones, such as estrogen or estrogen with progesterone (progestin in its synthetic form). ERT will be used going forward to prevent confusion with other hormone therapies. 7. King, M. The Medical Biochemistry Page. IU School of Medicine. http://themedicalbiochemistrypage.org/extracellularmatrix.html. Last modified June 11, 2008. 8. Bullough HF. Cyclical changes in the skin of the mouse during estrous cycle. J Endocrinol. 1943;3:280–287. 9. McConkey B, Fraser GM, Bligh AS, et al. Transparent skin and osteoporosis. Lancet. 1963;I:693–695. 10. Black MM, Bottoms E, Shuster S. Changes in skin collagen and thickness in endocrine disease. Eur J Clin Invest. 1970;1:127. 11. Meema HE, Sheppard RH, Rapoport A. Roentgenographic visualization and measurement of skin thickness and its diagnostic application in acromegaly. Radiology. 1964;82:411. 12. Rauramo L, Punnomen R. Wirkung einer oralen ostrogenotherapie mit ostriolsuccinat auf die Hautt kastrierter Frauen. Z Haut Geschl Kr. 1969;44:463–470. 13. Brincat M, Yuen AW, Studd JW, et al. Response of skin thickness and metacarpal index to estradiol therapy in postmenopausal women. Obstet Gynecol. Oct 1987;70(4):538–541. 14. Castelo-Branco C, Duran M, Gonzales-Merlo J. Skin collagen and bone changes related to age and hormone replacement therapy. Maturitas. 1992;14:113–119. 15. Affinito P, Palomba S, Sorrentino C, et al. Effects of postmenopausal hypoestrogenism on skin collagen. Maturitas. 1999;33:239–247. 16. Phillips TJ, Symons J, et al. Does hormone therapy improve agerelated skin changes in postmenopausal women? A randomized, double-blind, double-dummy, placebo-controlled multicenter study assessing the effects of norethindrone acetate and ethinyl estradiol in the improvement of mild to moderate age-related skin changes in postmenopausal women. J Am Acad Dermatol. Sept 2008;59(3):397–404, e3. 17. Haapasari K, Raudaskoski T, Kallioinen M, et al. Systemic therapy with estrogen or estrogen with progestin has no effect on skin collagen in postmenopausal women. Maturitas. 1997;27:153–162. 18. Maheux R, Naud F, Rioux M, et al. A randomized, double-blind, placebo-controlled study on the effect of conjugated estrogens on skin thickness. Am J Obstet Gynecol. 1994;170:642–649. 19. Varila E, Rantala I, Oikarinen A, et al. The effect of topical estradiol on skin collagen of postmenopausal women. Br J Obstet Gynaecol. Dec 1995;102(12):985–989. 20. Callens A, Valliant L, Lecomte P, et al. Does hormonal skin aging exist? A study of the influence of different hormone therapy regimens on the skin of postmenopausal women using non-invasive measurement techniques. Dermatology. 1996;193:289–294. 21. Sauerbronn AVD, Fonseca AM, Bagnoli VR, et al. The effects of systemic hormone replacement therapy on the skin of the postmenopausal women. Int J Gynecol Obstet. 2000;68:35–41. 22. Sator PG, Sator MO, Schmidt JB, et al. A prospective, randomized, double-blind, placebo-controlled study on the influence of a hormone replacement therapy on skin aging in postmenopausal women. Climacteric. 2007;10:320–334. 23. Sumino H, Ichikawa S, et al. Effects of aging, menopause, and hormone replacement therapy on forearm skin elasticity in women. J Am Geriatr Soc. 2004;52:945–949. 24. Savvas M, Laurent G. Type III collagen content in the skin of postmenopausal women receiving estradiol and testosterone implants. Br J Obstet Gynaecol. Feb 1993;100:154–156.
Biological Effects of Estrogen on Skin 25. Lee JY, Maibach HI. Corticosteroid skin atrophogenicity: assessment methods. Skin Res Technol. 1998;4:161–166. 26. Schmidt J, Binder M, Demschik G, et al. Treatment of skin aging with topical estrogens. Int J Dermatol. 1996;35:669–674. 27. Dunn L, Damesyn M, Moore A, et al. Does estrogen prevent skin aging? Results from the First National Health and Nutritional Examination Survey. Arch Dermatol. 1997;133:339–342. 28. Grosman N, Hridbey E, Schon J. The effect of estrogenic treatment on the acid mucopolysaccharide pattern in the skin of mice. Acta Pharmacol Toxicol. 1971;30:458–464. 29. Grosman N. Studies on the hyaluronic acid protein complex the molecular size of hyaluronic acid and the exchangeability of chloride in skin of mice before and after estrogen treatment. Acta Pharmacol Toxicol. 1973;33:201–208. 30. Pochi PE, Strauss JS, Downing D. Age related changes in sebaceous gland activity. J Invest Dermatol. 1979;73:108–111. 31. Pierard-Franchimont C, Letawe C, Goffin V, et al. Skin waterholding capacity and transdermal estrogen therapy for menopause: a pilot study. Maturitas. 1995;22:151–154. 32. Paquet F, Pierard-Franchimont C, Fuman I, et al. Sensitive skin at menopause; dew point and electrometric properties of the stratum corneum. Maturitas. 1998;28:221–227. 33. Sumino H, Ichikawa S, Abe M, et al. Effects of aging and postmenopausal hypoestrogenism on skin elasticity and bone mineral density in Japanese women. Endocrine J. 2004;51:159–164. 34. Contet-Audonneau JL, Jeanmaire C, Pauly G. A histological study of human wrinkle structures: comparison between sun-exposed areas of the face, with or without wrinkles, and sun-protected areas. Br J Dermatol. 1999;140:1038–1047. 35. Bosset S, Barre P, Chalon A, et al. Skin ageing: clinical and histopathologic study of permanent and reducible wrinkles. Eur J Dermatol. 2002;12(3):247–252. 36. Creidi P, Faivre B, Agache P, et al. Effect of a conjugated estrogen (Premarin) cream on ageing facial skin. A comparative study with a placebo cream. Maturitas. 1994;19:211–223. 37. Punnonen R, Vaajalahti P, Teisala K. Local estriol treatment improves the structure of elastic fibers in the skin of postmenopausal women. Ann Chir Gynaecol. 1987;202(Suppl):39–41. 38. Castelo-Branco C, Figueras F, Martinez de Osaba M, et al. Facial wrinkling in postmenopausal women. Effects of smoking status and hormone replacement therapy. Maturitas. 1998;29:75–86. 39. Brincat MP, Muscat Baron Y, Galea R. Estrogens and the skin. Climacteric. 2005;8:110–123.
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40. Ashcroft GS, Dodsworth J, van Boxtel E, et al. Estrogen accelerates cutaneous wound healing associated with an increase in TGF-b1 levels. Nat Med. 1997;3:1209–1215. 41. Ashcroft GS, Greenwell-Wild T, Horan MA, Wahl SM, Ferguson MW. Topical estrogen accelerates cutaneous wound healing in aged humans associated with an altered inflammatory response. Am J Pathol. 1999;155:1137–1146. 42. Ashcroft GS, Ashworth JJ. Potential role of estrogens in wound healing. Am J Clin Dermatol. 2003;4(11):737–743. 43. Whitby DJ, Ferguson MWJ. Immunohistochemical localization of growth factors in fetal wound healing. Dev Biol. 1991;147:207–215. 44. Shah M, Foreman DM, Ferguson MWJ. Control of scarring in adult wounds by neutralizing antibody to transforming growth factor B. Lancet. 1992;339:213–214. 45. Hu D, Hughes MA, Cherry GW. Topical tamoxifen – a potential therapeutic regime in treating excessive dermal scarring? Br J Plast Surg. Sept 1998;51(6):462–469. 46. Surazynski A, Jarzabek K, Haczynski J, Laudanski P, Palka J, Wolczynski S. Differential effects of estradiol and raloxifene on collagen biosynthesis in cultured human skin fibroblasts. Int J Mol Med. 2003;12:803–809. 47. Osborne K, Zhao HH, Fuqua SAW. Selective estrogen receptor modulators: structure, function, and clinical use. J Clin Oncol. 2000;18:3172–3186. 48. Hall GK, Phillips TJ. Skin and hormone therapy. Clin Obstet Gynecol. 2004;47(2):437–449. 49. Kanda N, Watanabe S. Regulatory roles of sex hormones in cutaneous biology and immunology. J Dermatol Sci. 2005;38(1):1–7. 50. Schmidt JB. Perspectives of estrogen treatment in skin aging. Exp Dermatol. 2005;14(2):156. 51. Thornton MJ. Estrogen functions in skin and skin appendages. Expert Opin Ther Targets. 2005;9(3):617–629. 52. Lynfield YL. Effect of pregnancy on the human hair cycle. J Invest Dermatol. 1960;35:323–327. 53. Whiting DA. Diagnosis of Alopecia. Current Concepts. Kalamazoo: A Scope Publication, The Upjohn, 1990. 54. Burger H. Hormone replacement therapy in the post-Women’s Health Initiative era. Report of a meeting held in Funchal, Madeira, February 24–25. Climacteric. 2003;6(Suppl 1):11–36.
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40 Biology of Stratum Corneum: Tape Stripping and Protein Quantification Ali Alikhan . Howard I. Maibach
Introduction Stratum corneum (SC) adhesive tape stripping has been utilized in the measurement of stratum corneum mass, barrier function, drug reservoir, and percutaneous penetration of topical substances. The process involves a methodical, relatively noninvasive layer-by-layer removal of the SC, which comprises the outermost epidermal cell layers. Complete SC removal may require over 70 tape strips [1, 2]. The quantity of SC harvested diminishes with each sequential strip, possibly due to increased SC cohesiveness in deeper layers. Thus, the mass of any single strip depends on the mass removed by the prior strip [3]. SC removal may rely on the interaction between the adhesive stripping force and the cohesive intercellular force [3].
Tape Stripping Studies Tape stripping was first devised in the 1940s, and examined by Pinkus in 1951. Pinkus demonstrated a remarkable burst of mitotic epidermal activity post-stripping, concluding that the lost horny layer is replaced by basal mitotic division [4]. The degree of hyperplasia correlates with the level and duration of barrier disruption [5]. Nevertheless, mitotic rate may remain five times greater than baseline six days after stripping [6]. Keratinocyte hyperproliferation may be a response to water barrier disruption or cytokine release secondary to epidermal injury [5, 6]. Adhesive stripping increases: epidermal lipid synthesis, lamellar body production/secretion in the stratum granulosum, epidermal DNA synthesis, epidermal cytokine production, dermal inflammation, and presence of TNF and IL-1a in skin [6]. Conversely, occlusion of stripped human skin via adhesive application suppresses mitotic activity; adhesive occlusion may provide artificial restoration of the lost barrier [6]. Similar experiments in mice do not support these findings [6]. The SC is essential to life, protecting the human body from desiccation and external penetration of deleterious agents. The SC is composed of a nucleated, keratin-rich
corneocytes embedded in an extracellular multilamellar lipid matrix organized into membrane-like bilayers; intercorneocyte communication occurs through desmosomes [7]. Many other SC models exist, but none of them fully integrate all aspects of the skin barrier function. The SC is thin, less than 20 mm thick, and composed of about 10–15 tightly stacked layers, depending on the location [8]. Ceramides, cholesterol, and free fatty acids comprise the lipid matrix of the SC, providing invaluable roles in the barrier structure and function [7]. Their synthesis is required for barrier homeostasis; as with DNA, a burst of lipid synthesis (due to synthesis of their rate-limiting enzymes) occurs following barrier perturbation [9]. Lipid levels decrease in aged human skin, possibly due to SC pH increases and subsequent lipid processing impairment; this is described further, below [7]. The SC provides the rate determining step for the passage of most molecules across skin [10]. Therefore, topical agent concentration within the SC is directly related to that in the epidermis and dermis, the typical target sites. Additionally, corneocytes and intercellular lipids are responsible for preventing insensible water loss [11]. The transepidermal water loss can be measured with an evaporimeter, and frequently used to assess skin barrier integrity [11]. Anatomically, regional SC variations in percutaneous drug absorption, lipid composition, TEWL measurements, mean thickness, and number of cell layers have been described. Despite its structural heterogeneity, each layer of SC equally contributes in preventing water loss [11]. In doing so, the SC behaves as a membrane compatible with Fick’s laws of passive diffusion [11]. TEWL increase as a function of tape strip number depends on the factors including: anatomical site, pressure, pressure duration, and tape removal rate [12]. Loffler et al. demonstrated that TEWL increased fastest on the forehead, followed by the back, and finally, the forearm [12]. These findings may be explained by the differences in SC thickness, differences in spontaneous desquamation (SC cohesion), and pressure resistance because of inherent viscoelasticity and type of tissue underlying the skin [12]. Rapid removal (vs. slow),
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_40, # Springer-Verlag Berlin Heidelberg 2010
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shorter pressure duration (2 vs. 10 s), and higher pressure (330 g cm 2 vs. 165 cm 2) all produced earlier TEWL increases [12]. A similar study by Breternitz et al. revealed the highest rise of TEWL on the cheek, compared to the back, upper arm, and forearm [13]. Interestingly, the cheek also demonstrated the greatest increase in SC hydration after stripping [13]. Breternitz et al. further established greater, earlier TEWL increase with higher pressure (7 N stamp vs. 2 N) and longer application (10 vs. 2 s) [13]. Moreover, using the thumb, stretching the skin, and utilizing a roller or stamp all result in varying quantities of harvested SC [13]. The use of thumb removed most of SC and produced the highest TEWL, even when compared with usage of a roller or skin stretching [13]. Occlusion of the test site prior to the stripping procedure resulted in higher TEWL values [13]. Occlusion results in water retention and degradation of the intercellular proteins [13]. In conclusion, reliable, reproducible results depend on standardization of the aforementioned variables. Kalia et al. found that initial tape strips removed thicker layers of the SC, relating this to decreased number of desmosomes closer to the skin surface [10]. Kalia et al. demonstrated decreased impedance with increasing depth achieved, theorizing that removal of the upper corneocyte layers and lipid matrix diminishes structural opposition to ion flow, facilitating ion transport [10]. In addition, TEWL increased disproportionally with later tape strips; removing only the upper SC layers was insufficient to significantly enhance the water loss [10]. Removal of 6–8 mm of SC (deeper layers) typically resulted in significant TEWL increases [10]. Removal of the outermost layers affected impedance more than TEWL, with a 40% decrease in impedance after removal of only 4 mm of SC. Nonetheless, a correlation between TEWL increase and impedance decrease was observed. Upon completion of the tape stripping experiment, full return to the basal values of impedance occurred after 3 days, while TEWL recovery time was 5–6 days [10]. External layers, more crucial in impedance, are formed prior to deep compact layers [10]. The aforementioned findings suggest a gradation in water-regulating ability within the SC, with the deepest layers most responsible for controlling water flux [10]. However, via simple mathematical deduction, these results, in fact, support a Fickian model [10]. Though structurally heterogeneous and complex, the SC behaves as a homogenous barrier to water in vivo [10]. The water transport route may be homogeneous throughout SC, with each layer contributing equally to the barrier [10]. The best fit curve plotting experimental values of TEWL
as a function of tape stripping frequency closely resembled a theoretical curve based on Fick’s first law of diffusion [14]. The first half of the theoretical curve fit the actual curve; in the second half, experimental data show slightly higher TEWL values than Fick’s theoretical values [14]. The authors of the study offer plausible explanations for this discrepancy [14]. In contrast to most studies, Schwindt et al. demonstrated that quantity of harvested SC was constant with each strip in a given anatomical site and volunteer [11]. Schwindt et al. found a linear relationship (in all anatomical sites) between 1/TEWL and the total mass of removed SC, further establishing that the SC acts as a Fickian membrane for steady state water diffusion [11]. It also appears that intercellular lipids, not corneocytes, are the determining factor for SC water diffusion [11]. This linear relationship was also described by another group, plotting 1/TEWL as a function of SC thickness (13 subjects examined) [15]. > Table 40.1 summarizes the results from three studies quantifying SC thickness. Tape construction influences outcome [13]. Three brands of adhesive tapes, utilized in vivo, displayed statistically equivalent mean water diffusion coefficients, SC permeability, and SC mass/thickness removal [3]. After 40 strips, however, a proprietary adhesive stripped the most, while a rayon adhesive stripped the least [3]. TEWL increased significantly as deeper SC layers were reached with proprietary and polyethylene adhesives, but not with rayon tape [3]. Tape properties, subject properties, or a combination may account for variation
. Table 40.1 Calculations of SC thickness in vivo in man
Authors
No. of Anatomical subjects site
No. of strips
Mean total SC thickness (mm)
Kalia et al. [10]
3
Forearm
22–28
12.7 3.3
Schwindt et al. [11]
6
Lower back
Up to 35
11.2
Pirot et al. [15]
13
Abdomen
Up to 35
7.7
Thigh
Up to 35
13.1
Forearm (ventral)
Up to 35
12.3 3.5
Forearm (ventral)
15
12.6 5.3
Thickness appears to be a function of anatomical site
Biology of Stratum Corneum: Tape Stripping and Protein Quantification
in barrier disruptive properties. Variation may also be accounted for by unique adhesive systems; adhesives of different tape brands may bind similarly to cellular SC, but differently to extracellular components of the SC barrier. These extracellular components (e.g., free fatty acids, ceramides, and lipids) are essential to barrier function. Furthermore, apparently 5–7 mm of SC removal resulted in significant TEWL elevations, a depth unobtainable by the rayon tape (> Table 40.2) [3]. This implies that structural elements of the water barrier may not be homogeneously distributed. In some subjects, neither the proprietary adhesive nor the polyethylene adhesive disrupted the water barrier; these individuals experienced no barrier disruption at any of six tested sites, suggesting variation of water barrier disruption to be a function of the individual. Demonstrating that removal of the same amount of SC from different individuals does not result in similar increases in TEWL, Kalia et al. asked whether this variation was secondary to inter-individual differences in intact membrane thickness [16]. Kalia et al. demonstrated that once inter-individual differences in the thickness of the intact SC are corrected for (by normalizing the SC thickness removed with respect to calculated total SC thickness), the same degree of barrier disruption induces the same increase in TEWL in each individual [16]. Stated differently, removal of the same percentage of SC in two individuals results in equivalent barrier disruption. TEWL rises considerably only after about 75% of the SC has been removed, presenting a very consistent barrier to water loss in the healthy human population [16].
. Table 40.2 Relationship between protein removal and TEWL, from Bashir et al. [3]
Tape type Proprietary
No. Location of (forearm) strips
TEWL (g m 1 h 1) 30.33
Dorsal
40
8.10
Ventral
40
5.83
30.80
40
7.25
31.98
Ventral
40
4.96
30.83
Dorsal
40
4.99
13.4
Ventral
40
2.99
11.95
Polyethylene Dorsal Rayon
Mean thickness removed (mg)
Note, there are significant differences in TEWL and mean thickness removed depending on tape construction. The dorsal forearm, in all cases, had greater SC thickness removed than the ventral forearm
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Tape Stripping and Aging Aged skin demonstrates increased susceptibility to the xerosis, exogenous, and environmental insults, and diminished ability to recover from these insults, indicating a suboptimal epidermal barrier. It is believed that no definitive studies have compared aged vs. normal SC thickness; nonetheless, some authors believe aged SC to be thicker, with decreased lipid content and deficient water-binding capacity [8]. TEWL is decreased in the aged, as is topical absorption [8]. The aging barrier was elegantly examined by Ghadially et al.; results are summarized below. Aged humans (>80 years) have prolonged barrier recovery rates after tape stripping or acetone application compared to control subjects (20–30 years) [17]. 24 h after acetone treatment, 50% recovery occurred in control subjects compared to 15% in aged subjects [17]. Photoaging, in combination with this chronologic aging, may further delay recovery [9]. Furthermore, delays in SC lipid reappearance after barrier disruption have been described in aged murine epidermis [17]. Additionally, tape stripping studies have revealed decreased cohesiveness in aged skin [9]. In fact, barrier perturbation (TEWL 20 g m 2 h 1) occurred after 18 2 strippings in aged skin versus 31 5 strippings in control skin [17]. Fortunately, topical lipid formulations, containing predominantly cholesterol, may accelerate barrier recovery in aged human skin [18]. The above findings may be explained by reduced delivery of secreted lipids to the epidermal surface in the elderly. There is a global diminution ( 30%) of ceramide, cholesterol, and free fatty acid contents in the aged murine skin [17]. This reduction could be due to the decreased production and/or increased destruction; cytokines (e.g., IL-1a) and growth factors may play a role [9]. Additionally, decreased secretion of lamellar body contents (at stratum granulosum-stratum corneum interface) with fewer extracellular lamellar bilayers (at stratum corneum interstices) contributes to a more porous extracellular SC matrix [17]. Ghadially et al. further examined the effect of lipids on SC barrier function [19]. As described previously, SC of aged mice displays decreased lipid content and extracellular bilayers. This may result in impaired barrier recovery after a tape stripping insult (18.7 vs. 60.8% recovery by 24 h in aged vs. young mice). Upon further examination, Ghadially et al. determined that cholesterol synthesis is decreased significantly under basal conditions. Furthermore, sterologenesis fails to reach absolute levels obtained in young epidermis following tape stripping perturbation.
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Biology of Stratum Corneum: Tape Stripping and Protein Quantification
A 40% decrease in activity of HMG-CoA reductase, the rate-limiting enzyme in sterologenesis, was observed under basal conditions in aged mice. Despite a greater than 100% increase in HMG-CoA reductase activity after barrier perturbation in aged mice, absolute levels did not attain those reached in treated, young epidermis. Ghadially et al. also supplemented aged murine SC with an equimolar mixture of SC physiological lipids (cholesterol:ceramide:linoleic acid:palmitic acid) or cholesterol alone [19]. Either mixture applied once enhanced the recovery after barrier disruption. Additionally, after four applications of either mixture, electron microscopy demonstrated repletion of extracellular spaces with normal lamellar bilayer structures. Further work examining the role of aging on the SC remains to be done. Tape stripping and TEWL studies of aged skin are currently underway.
Protein Quantification After harvesting of SC onto adhesives is complete, protein can be measured via several methods. For decades, weighing (gravimetry) was the preferred method, despite its inherent inconvenience (weighing before and after stripping under constant hydration conditions). Additionally, results were subjected to inflation secondary to absorption of exogenous (topically applied) or endogenous (sebum, sweat, and interstitial fluid) substances within the SC. Initial strips were most affected by this absorption. One decade ago, a novel colorimetric method was developed and validated by Dreher et al. [20]. This colorimetric method relies on a protein assay similar to one developed by Lowry et al. over half a century ago. Lowry’s method involved measurement of protein with a folin phenol reagent after alkaline copper treatment [21]. It was demonstrated to be simple, sensitive, specific, and easily adaptable to small scale analyses, making it suitable for measurement of miniscule absolute protein amounts [21]. Dreher’s method relies on spectrophotometry and colorimetry, based on the calibration of stained SC proteins to the corneocyte mass [22]. Drawbacks include time-consuming preparation of tape strips with necessary destruction of the original strips. The Bradford dye reaction, which relies on Coomassie Brilliant Blue G-250 dye, is similar to Dreher’s method. The dye binds protein, resulting in ionic and hydrophobic reactions, with a spectral shift from reddish-brown to blue. Maximal absorption for the bound form of the dye is 595 nm, the optimal wavelength for colorimetric measurement once the reaction has occurred. Despite
disadvantages (e.g., serial dilutions), it is a fast and generally reliable method for protein quantification. Dreher’s colorimetric method has been successfully adapted to 96-well microplates, effectively shortening analysis time [23]. Note that limited areas of adhesive tape are not predictive of SC removal on the entire tape [23]. Alternatively stated, SC distribution on tape is not homogeneous [23]. A pivotal study examined direct spectroscopic SC protein quantification via absorption in the visible range (595 and 600 nm), with and without staining of corneocyte aggregates, and the UV range (278 nm) [24]. Correlation coefficients R2 were 0.71 and 0.74, respectively. The results demonstrated weak SC protein absorption with immense light scattering [24]. The Coomassie brilliant blue protein coloring did not increase light absorption by SC proteins, and thus, could not decrease the interference secondary to light scattering [24]. The absorption techniques utilized in this study cannot accurately predict corneocyte aggregate quantity. Latter studies utilizing wavelengths of 430 nm have established optical spectroscopy in the visible range as a sensitive and reproducible method of protein quantification [2]. Absorbance in this range depends exclusively on quantity of corneocyte aggregates, and adequately reflects SC mass [2]. Corneocyte aggregates, adhering to tape strips, decrease transmission of visible light by scattering, reflection, and diffraction. The resulting pseudoabsorption has been successfully correlated with mass of removed SC particles [2]. Absorbance measurement allows facile determination of absolute mass from corneocyte aggregates harvested via tape stripping. Topically applied substances do not interfere with the spectroscopic measurements as they do with gravimetric measurements, explaining mass differences in the most superficial strips (when compared with gravimetry) [25]. Practically comparing spectrally measured quantity (absorbance) with corneocyte aggregate weight requires correction for: topical applications in upper SC layers, interstitial fluid in deeper SC layers, the ‘‘stack effect’’ which decreases absorbance, and the tape stripping procedure itself (e.g., nonhomogeneous removal of tape or incomplete tape contact with skin) [2]. Once these factors are corrected for (primarily by excluding analysis of the most superficial and deep strips), R2 = 0.93, demonstrating proportionality between quantification methods [2]. A multicenter study involving 24 subjects found a correlation coefficient of R2 = 0.94 when comparing UV/VIS spectroscopy (430 nm) with conventional weight determination [25]. Superficial (first five) and deep (19–23) strips were excluded on the basis of weight-enhancement;
Biology of Stratum Corneum: Tape Stripping and Protein Quantification
application on an oil-water emulsion (part of the study) inflated superficial strip weight, and intrinsic interstitial fluid increased deep strip weight [25]. Nonhomogeneous strips and those subjected to handling errors were excluded [25]. Only 66% of total strips were utilized to determine the correlation coefficient [25]. Weigmann et al. explain that pseudo-absorption/weight correlation can be extrapolated to the deepest layers of the SC [25]. A recent study demonstrated strong correlation (R2 = 0.92 and R2 = 0.95) between pseudo-absorption at 430 nm and both protein absorption at 278 nm and absorption of Trypan blue-stained proteins at 652 nm [22]. However, protein absorption at 278 nm was characterized by a weak band, implying application limited to tape strips with high amounts of corneocytes [22]. Mass determination based on the UV absorption is further limited by the potential superpositioning of strong absorption bands from exogenous substances and/or tape components in the same spectral range. Unlike the previous study, correlation was described using all the tape strips (superficial and deep), regardless of adherent exogenous or endogenous components [22]. Lademann et al. tested an inexpensive, easily reproducible optical device (‘‘corneocyte density analyzer’’), based on a slide projector, which also measures corneocyte pseudoabsorption at 430 nm [1]. When compared with standard UV-visible spectrometric measurements, a correlation factor of R2 = 0.95 was demonstrated [1]. The device may simplify calculation of removed SC, without messy chemistry or an expensive spectrometer; it includes a mechanical autofeed system, well suited for the handling of tape strips [1].
Colorimetric Bioassay of Keratolytic Efficacy The desquamating effects of three keratolytics are presented in table-format (> Table 40.3) using the data
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obtained from colorimetric protein assays described by Dreher et al. (mentioned above) [20]. The process begins with cutaneous application of the agent; the agent is placed on a patch and taped onto the subject’s skin for a predetermined number of hours. After this period, placement and removal of tape strips (number varies by study) onto the site of topical treatment are performed. The assay involves immersion and shaking of SC adhering tapes in sodium hydroxide solution resulting in extraction of the soluble SC protein fraction. The solution, now containing SC protein, is neutralized with hydrogen chloride, as the assay is ineffective under strongly alkaline conditions. The protein assay is performed using the Bio-Rad Detergent Compatible Protein Assay Kit and following the prescribed microassay procedure. This assay is similar to the Lowry assay, and is based on the reaction of protein with an alkaline copper tartrate solution and Folin reagent. Finally, absorbance at 750 nm is measured using a Hitachi U-2001 UV-vis Spectrophotometer. This method allows for quantification of microgram amounts of SC, diminishing confounding factors, namely vehicle and water uptake by the SC. The protein measured using the assay described can be compared amongst groups, with statistical analysis allowing determination of strong and weak keratolytics. SC removal via tape stripping in treatment and control groups is attributable to keratolytic mechanisms, which loosen SC cohesion. The disintegrated SC is subsequently collected by the adhesive. In the first keratolytic bioassay using this technique, salicylic acid was examined [26]. Keratolytic efficacy of salicylic acid was determined as a function of pH. The test preparations were: aqueous vehicle control of pH 7.4, 2% SA aqueous solution of pH 3.3, 2% SA aqueous solution of pH 3.3 with menthol, and 2% SA aqueous solution of pH 6.95 [26]. A statistically significant mass of SC was removed after 6 h and 20 tape strips in all three
. Table 40.3 Studies using colorimetric protein assay to measure keratolytic potential Authors
Drug
Result
Bashir et al. (2005)
Aqueous solution 2% Salicylic Acid – 3 formulations
Statistically significant mass of SC removed after 6 h and 20 tape strips in all three experimental groups (salicylic acid pH 3.3, salicylic acid pH 3.3 w/ menthol, salicylic acid pH 6.95) compared to vehicle, untreated, and untreated but occluded groups.
Waller et al. (2006)
Aqueous solutions of 0.05 % all-trans RA, 2% BPO, and 2% SA
Statistically significant mass of SC removed after 6 h and 25 tape strips in all three experimental groups compare to vehicle, untreated, and occluded groups. The first 10 tape strips from SA group removed more protein than the other groups; at 10–15 strips, treatments were comparable; at 16–25 strips, protein removed from BPO sites was greatest.
All agents tested demonstrated significant efficacy in SC removal. SA had superior superficial removal, while BP had superior deep removal
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Biology of Stratum Corneum: Tape Stripping and Protein Quantification
experimental groups compared to vehicle, untreated, and untreated but occluded groups [26]. However, after 10 strips, the SA pH 3.3 solution with menthol and the SA pH 6.95 solution removed significantly more SC than any other group, including the SA pH 3.3 solution [26]. These data suggest that a neutral preparation of SA results in a pronounced keratolytic effect. Moreover, the neutral preparation was associated with the least skin irritation among treatment groups [26]. This finding differs from that of a previous study, which demonstrated superior SA skin penetration in an acidic solution compared to neutral solution [27]. In the second bioassay using the aforementioned technique, salicylic acid, benzoyl peroxide (BPO), and retinoic acid were examined [28]. The test preparations were: 0.05% all-trans retinoic acid, 2% salicylic acid at pH 6.95, 2% BPO, vehicle, untreated skin, and occluded but untreated skin [28]. After 3 h of treatment, only BPO treatment removed significantly more SC on 25 strips than untreated skin, while the other treatments did not achieve statistical significance [28]. At 3 h, SA had greater SC amounts removed in the first 10 (superficial) strips, while deeper strips (11–25) demonstrated BPO to have the greatest SC removal [28]. Statistically significant masses of SC were removed after 6h and 25 tape strips in all three experimental groups when compared to vehicle, untreated, and occluded groups [28]. At 6 h, the first 10 tape strips from the SA group removed more protein than the other groups; at 10–15 strips, all treatments were comparable; at 16–25 strips, BPO removed the most protein [28]. These in vivo human results indicate that all treatments tested are effective keratolytics, which may account for their effectiveness against acne vulgaris. Furthermore, it appears that salicylic acid may be a more suitable treatment for mild, superficial acne while BPO may be optimal for deeper, inflammatory acne. BPO’s ability to loosen SC at deeper levels complements its antimicrobial/ anti-inflammatory properties, resulting in an effective anti-inflammatory agent for papulo-pustular acne. Additionally, BPO appears to be effective even with short-term administration. RA had inferior SC disruption at 3 h but significant disruption at 6 h, indicating time-dependent keratolytic effects, consistent with its complex nuclear receptor interactions and alteration of gene transcription.
Conclusion Taken together, the SC is beginning to reveal some of its secrets. Much remains to be done.
Cross-references > Corneocyte
Size and Cell Renewal: Effects of Aging and Sex Harmones > Stratum Corneum Cell Layers > The Stratum Corneum and Aging
References 1. Lademann J, Ilgevicius A, Zurbau O, Liess HD, Schanzer S, Weigmann HJ, Antoniou C, Pelchrzim RV, Sterry W. Penetration studies of topically applied substances: optical determination of the amount of stratum corneum removed by tape stripping. J Biomed Opt. 2006; 11(5):054026. 2. Weigmann H, Lademann J, Meffert H, Schaefer H, Sterry W. Determination of the horny layer profile by tape stripping in combination with optical spectroscopy in the visible range as a prerequisite to quantify percutaneous absorption. Skin Pharmacol Appl Skin Physiol. 1999;12(1–2):34–45. 3. Bashir SJ, Chew AL, Anigbogu A, Dreher F, Maibach HI. Physical and physiological effects of stratum corneum tape stripping. Skin Res Technol. 2001;7(1):40–48. 4. Pinkus, H. Examination of the epidermis by the strip method of removing horny layers. I. Observations on thickness of the horny layer, and on mitotic activity after stripping. J Invest Dermatol. 1951;16(6):383–386. 5. Denda M, Wood LC, Emami S, Calhoun C, Brown BE, Elias PM, Feingold KR. The epidermal hyperplasia associated with repeated barrier disruption by acetone treatment or tape stripping cannot be attributed to increased water loss. Arch Dermatol Res. 1996;288 (5–6):230–238. 6. Fisher LB, Maibach HI. Physical occlusion controlling epidermal mitosis. J Invest Dermatol. 1972;59(1):106–108. 7. Jungersted JM, Hellgren LI, Jemec GB, Agner T. Lipids and skin barrier function – a clinical perspective. Contact Dermatitis. 2008;58(5):255–262. 8. Tagami H. Functional characteristics of the stratum corneum in photoaged skin in comparison with those found in intrinsic aging. Arch Dermatol Res. 2008;300(Suppl 1):1–6. 9. Elias PM, Ghadially R. The aged epidermal permeability barrier: basis for functional abnormalities. Clin Geriatr Med. 2002;18 (1):103–120, vii. 10. Kalia YN, Pirot F, Guy RH. Homogeneous transport in a heterogeneous membrane: water diffusion across human stratum corneum in vivo. Biophys J. 1996;71(5):2692–2700. 11. Schwindt DA, Wilhelm KP, Maibach HI. Water diffusion characteristics of human stratum corneum at different anatomical sites in vivo. J Invest Dermatol. 1998;111(3):385–389. 12. Loffler H, Dreher F, Maibach HI. Stratum corneum adhesive tape stripping: influence of anatomical site, application pressure, duration and removal. Br J Dermatol. 2004;151(4):746–752. 13. Breternitz M, Flach M, Prassler J, Elsner P, Fluhr JW. Acute barrier disruption by adhesive tapes is influenced by pressure, time and anatomical location: integrity and cohesion assessed by sequential tape stripping. A randomized, controlled study. Br J Dermatol. 2007;156(2):231–240.
Biology of Stratum Corneum: Tape Stripping and Protein Quantification 14. van der Valk PG, Maibach HI. A functional study of the skin barrier to evaporative water loss by means of repeated cellophane-tape stripping. Clin Exp Dermatol. 1990;15(3):180–182. 15. Pirot F, Berardesca E, Kalia YN, Singh M, Maibach HI, Guy RH. Stratum corneum thickness and apparent water diffusivity: facile and noninvasive quantitation in vivo. Pharm Res. 1998;15 (3):492–494. 16. Kalia YN, Alberti I, Sekkat N, Curdy C, Naik A, Guy RH. Normalization of stratum corneum barrier function and transepidermal water loss in vivo. Pharm Res. 2000;17(9):1148–1150. 17. Ghadially R, Brown BE, Sequeira-Martin SM, Feingold KR, Elias PM. The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model. J Clin Invest. 1995;95(5):2281–2290. 18. Zettersten EM, et al. Optimal ratios of topical stratum corneum lipids improve barrier recovery in chronologically aged skin. J Am Acad Dermatol. 1997;37(3 Pt 1):403–408. 19. Ghadially R, Brown BE, Hanley K, Reed JT, Feingold KR, Elias PM. Decreased epidermal lipid synthesis accounts for altered barrier function in aged mice. J Invest Dermatol. 1996;106(5):1064–1069. 20. Dreher F, Arens A, Hostynek JJ, Mudumba S, Ademola J, Maibach HI. Colorimetric method for quantifying human Stratum corneum removed by adhesive-tape stripping. Acta Derm Venereol. 1998;78 (3):186–189. 21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193 (1):265–275.
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22. Lindemann U, Weigmann HJ, Schaefer H, Sterry W, Lademann J. Evaluation of the pseudo-absorption method to quantify human stratum corneum removed by tape stripping using protein absorption. Skin Pharmacol Appl Skin Physiol. 2003;16(4):228–236. 23. Dreher F, Modjtahedi BS, Modjtahedi SP, Maibach HI. Quantification of stratum corneum removal by adhesive tape stripping by total protein assay in 96-well microplates. Skin Res Technol. 2005;11 (2):97–101. 24. Marttin E, Neelissen-Subnel MT, De Haan FH, Bodde HE. A critical comparison of methods to quantify stratum corneum removed by tape stripping. Skin Pharmacol. 1996;9(1):69–77. 25. Weigmann HJ. UV/VIS absorbance allows rapid, accurate, and reproducible mass determination of corneocytes removed by tape stripping. Skin Pharmacol Appl Skin Physiol. 2003;16(4):217–227. 26. Bashir SJ, Dreher F, Chew AL, Zhai H, Levin C, Stern R, Maibach HI. Cutaneous bioassay of salicylic acid as a keratolytic. Int J Pharm. 2005;292(1–2):187–194. 27. Leveque N, Makki S, Hadgraft J, Humbert P. Comparison of Franz cells and microdialysis for assessing salicylic acid penetration through human skin. Int J Pharm. 2004;269(2):323–328. 28. Waller JM, Dreher F, Behnam S, Ford C, Lee C, Tiet T, Weinstein GD, Maibach HI. ‘‘Keratolytic’’ properties of benzoyl peroxide and retinoic acid resemble salicylic acid in man. Skin Pharmacol Physiol. 2006;19(5):283–289.
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14 Buffering Capacity Considerations in the Elderly Jacquelyn Levin . Howard I. Maibach
Introduction The acidic character of skin was first mentioned by Heuss [1] and later by Schade and Marchionini [2] who introduced the term ‘‘acid mantle’’ for skin’s acidic outer surface pH. It has been recognized as playing a crucial role in permeability barrier homeostasis, skin integrity/cohesion and immune function [3–5]. Given this, it is important for skin to be able to resist acidic/alkaline aggression to some extent (i.e., have buffering capacity) [6]. The pH of skin increases and the ability to buffer the change in skin pH decreases with age [7]. This increase in pH and decreased buffering capacity in elderly skin results in impaired barrier homeostasis and skin integrity/cohesion, increased likelihood for skin infection, and increased sensitivity/irritation to topically applied products [8]. The chapter briefly reviews the basic science of pH and buffering capacity and the deleterious effects of increased pH in the skin of the elderly. The decrease in buffering capacity in elderly skin will be discussed, firstly by discerning which components of the stratum corneum are most likely responsible for the buffering capacity in skin of all ages and secondly by reviewing the physiologic changes of the stratum corneum that may contribute to the decrease in the buffering capacity detected clinically in elderly skin.
Defining and Measuring the pH and Buffering Capacity of Skin When dilute aqueous acid or alkaline solutions come into contact with skin, the change in pH is generally temporary and the original skin pH (a measure of the hydronium ion concentration) is rapidly restored, indicating that skin has significant buffering capacity. A buffer is a chemical system that can limit changes in pH when an acid or a base is added. Buffer solutions consist of a weak acid and its conjugate base. The system has its optimum buffering capacity when about 50% of
buffer is dissociated, or in other words at a pH approximately equal to its pKa [6, 9]. The pKa is the negative of the common logarithm of the acid dissociation constant (Ka) and is a measure for the strength of the acid. The buffer capacity is further dependent on the concentration of the system. An acid/alkali aggression test is one way to measure the acid/alkali resistance (i.e., buffering capacity) of skin. Alkali/Acidic resistance tests were commonly used in the 1960s to detect workers that may likely develop occupational diseases in certain chemical work environments [6]. A mild variation of the alkali/acidic resistance tests, also called acid/alkali neutralization test, assesses how quickly the skin is able to buffer applied acids/bases without the occurrence of skin corrosion. Repetitive applications of acid or base demonstrate that the skin’s buffering capacity is limited and may be overcome, as illustrated by the long time required for neutralization [10–13].
Effect of Increase in pH on Elderly Skin Function and Defenses In a multicenter study on measurement of the natural pH of the skin surface, the values of skin surface pH were 4.9 (arithmetic mean) with a 95% confidence interval between 4.1 and 5.8 [9]. Ideal acidity for the stratum corneum is a pH of approximately 5.4 [14]. It is well known that an increased skin pH is detected in the elderly skin starting anywhere from age 50 to 80 years [14–18]. Most likely this decreased acidity is due to less efficient mechanisms for skin acidification and more specifically decreased NA + /H + antiporter (NHE1) expression. The NHE1 is one of three highly studied mechanisms for maintaining skin acidity and is assumed to be the predominate mechanism for maintaining skin acidity [18]. Elevation of the skin pH in the elderly alters multiple functions. Those discussed here include impairment of permeability barrier homeostasis, decreased skin integrity/cohesion, and increased susceptibility for microbial infection.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_14, # Springer-Verlag Berlin Heidelberg 2010
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Impaired Permeability Barrier Homeostasis An acidic pH is critical for permeability barrier homeostasis, in part because of two key lipid-processing enzymes, B-glucocerebrosidase and acid sphingomyelinase which generate a family of ceramides from glucosylceramide and sphingomyelin precursors and exhibit low pH optima [19]. An increased skin pH results in defective lipid processing and delayed maturation of lamellar membranes [18]. These lipids form multi-lamellar sheets amidst the intracellular spaces of the stratum corneum critical to the stratum corneum’s mechanical and cohesive properties, enabling it to function as an effective water barrier [18]. This delayed barrier function allows easier penetration of topically applied products and delays barrier recovery after injury or insult to the skin [18, 19].
Decreased Skin Integrity and Cohesion An acidic pH also clearly promotes stratum corneum integrity and cohesion. In a neutral pH environment there is an enhanced tendency for the stratum corneum to be removed by tape stripping (integrity) as well as increased amount of protein removed per stripping (cohesion) [18, 20]. The impaired stratum corneum integrity/cohesion is due to pH dependent activation of serine proteases which exhibit neutral pH optima [21]. Serine proteases become activated in the increased pH of elderly skin and lead to the premature degradation of corneodesmosomes and hence increased desquamation [18, 19, 22].
Increased Susceptibility for Skin Infections The acidic pH of the stratum corneum restricts colonization by pathogenic flora and encourages persistence of normal microbial flora. Pertinently elderly skin, intertriginous areas, and chronically inflamed skin display an increased skin pH [2] and hence reduce resistance to pathogens [14]. In summary, elderly skin commonly has abnormalities in stratum corneum integrity/cohesion, permeability barrier homeostasis, and immune function due to increased skin pH. These abnormalities are attributable to the pH mediated increase in serine protease mediated degradation of corneodesmosomes, defect in lipid processing, and decrease in antibacterial activity, respectively.
Effect on Buffering Capacity of Skin in the Elderly and Elderly Skin Both the skin surface pH and reduced buffering capacity have been documented for skin of elderly. The reduced buffering capacity contributes to the increased sensitivity of skin to contact irritants and cleansing procedures [8]. The next section focuses on the aggression tests aimed at discerning which components of the epidermis are responsible for skin’s buffering capacity.
Free Fatty Acids/Sebum Early experimentation hypothesized that the sebum contributes to the buffering capacity of skin in two ways: first, it protects the epidermis against the influence of alkali by slowing down the exposure and penetration of acids or alkalis applied to the skin [23–25] and second, the fatty acids in sebum may act as buffer system [26, 27]. Later Lincke et al. [28] refuted the second hypothesis by demonstrating that the sebum had no relevant acid and a negligible alkali buffering capacity around pH 9. Further challenging the hypothesis, a quicker neutralization was observed on delipidized skin than untreated skin [23, 26]. Vermeer concluded similarly when comparing the neutralization on soles and forearm with and without sebum removal respectively [27]. However, when comparing these different skin regions, differences in sebum content may have also contributed to the observed effect. Vermeer [25] and Neuhaus [29] believed that the increased rate of neutralization after sebum removal may have been due to increased amounts of carbon dioxide (CO2) diffusion. This theory, discussed later in detail, is generally not accepted and also not clearly substantiated. After lipid removal, skin starts to increase acid production which may account for the faster neutralization. The same investigators also found that the increase in neutralization after lipid removal is temporary and limited to the first few minutes, which is probably related to the activity of sebaceous glands to produce relevant amounts of sebum. Due to the negligible buffering capacity of sebum and to standardize experimentation (limit inter- and intraindividual variability), today most neutralization experiments are performed after cleansing the skin with solvents which remove most of the sebum including fatty acids.
Buffering Capacity Considerations in the Elderly
Epidermal Water-Soluble Constituents
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Vermeer et al. [27] first demonstrated the importance of water-soluble constituents to the skin’s buffering ability. Water soaked skin, where the water-soluble constituents were extracted, demonstrated a significantly reduced neutralization capacity indicating that water-soluble substance constituent(s) of the skin are major contributors to the buffering capacity [12, 30, 31]. The significance of water-soluble constituents of the epidermis to the buffering capacity of skin further supports the theory of minimal contribution from the sebum of skin due to its lipid soluble nature [27].
in the water-soluble portion of the epidermis. However, the AA composition of keratin [41, 42] does not correspond with the composition of free AA found in the water-soluble portion of the stratum corneum [34], which implies that keratin is not a major contributor to the pool of free AA. Despite little evidence of keratin’s role in the buffering capacity, a modifying action of keratin is assumed [28]. Without an intact keratin layer, neither a physiological surface pH nor normal neutralization capacity can be maintained [43]. Further research remains to be conducted to determine keratin’s role in the buffering capacity of the epidermis.
Sweat
CO2
Eccrine sweat initially accelerates the neutralization of alkalis [10–13, 27, 30, 32, 33]. Spier and Pasher [34] suggest that the main buffering agents of sweat are lactic acid and amino acids. The lactic acid-lactate system in sweat has a highly efficient buffering capacity between pH 4 and 5 [24]. However, it has not been completely demonstrated that lactic acid is the main buffering agent in sweat or at the surface of the skin. Conversely, the contribution of amino acids (AA) to the buffering capacity of sweat and of the horny layer surface has been investigated thoroughly [27, 30, 33]. By comparing sweating and non-sweating persons, Vermeer [27] found that AA play a significant role in neutralization during the first 5 min while lactic acid does not. This confirms that AAs are key elements contributing to the buffering capacity of skin.
Little is known about the role of CO2/HCO3 participating in skin’s buffering capacity. Burckhardt’s studies were the first to suggest that the CO2 diffusing from the epidermal layer may be responsible for neutralizing alkali in contact with skin [10–13]. He demonstrated that when a 5 min alkali-neutralization experiment is repeated subsequently several times on the same skin area, the neutralization times became longer and finally reach an approximately constant time. He suggested that the shorter neutralization times at the beginning were due to acids present on the skin surface rapidly neutralizing the alkali. He further suggested that after successive alkali exposure, the endogenous acids were no longer present on the skin surface resulting in longer neutralization times and diffusing carbon dioxide would take over the role of neutralizing the alkali. At this time, Burckhardt’s hypothesis of the role of carbon dioxide as a buffering agent was accepted by others despite the rather weak experimental evidence [28, 36, 44, 45]. The decreased neutralization time after lipid removal of the skin surface with the help of soaps or neutral detergents was believed by Burckhardt and others to be the consequence of greater diffusion of CO2 although this has never been quantified [24, 44, 45]. It was also postulated that a more hydrated stratum corneum retains a greater amount of CO2 by limiting its diffusion. Therefore a moderate hydration level was regarded better for effective alkali neutralization; however, this has also never been analyzed in further detail [45]. Knowing that several authors considered CO2 a relevant contributor in alkali neutralization without having quantitative data to sustain their hypothesis, Vermeer et al. [19] demonstrated that CO2 is unlikely of great
Keratin The contribution of keratin to the buffering capacity of skin remains questionable. Keratin is an amphoteric protein with the ability to neutralize acids and alkalis in vitro [10– 13, 28, 35–37] and hence may participate in skin’s buffering capacity. Scales scraped from normal skin bind small amounts of alkali in vitro [38, 39]. However, Vermeer and coworkers showed that water-soluble constituents of the epidermis participate more in skin’s buffering capacity than the insoluble constituents of the skin such as keratin. While insoluble keratin filaments on the skin may have only little buffering capacity [27, 40], keratin hydrolysates (free amino acids) may contribute to the free AAs
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importance for alkali neutralization on skin. His experiment was focused on the first minutes of the neutralization process in contrast to the previous experiments mentioned [29, 36, 44, 45], which paid attention to the later neutralization process. For example, Piper [36] analyzed the neutralization process for up to 1 h and concluded that, for the first ½ hour, alkalis are neutralized on the skin by the skin’s own amphoteric substances (such as amino acids) but that in the second half-hour diffusing carbon dioxide takes over. Piper’s conclusions are not necessarily contradictory to the results obtained by Vermeer above and may actually be in agreement. According to Piper, ‘‘the longer the contact between skin and alkali, the greater the importance of CO2.’’ This statement is supported by the recent discoveries of relatively low level of carbon dioxide production in the epidermis and the limited activity of the Kreb’s Cycle, suggesting that a minimal amount of CO2 would be available for neutralization [41]. It seems likely that CO2 does not significantly contribute in the alkali neutralization process. Further studies should further help to clarify the relevance of CO2 in skin’s buffering capacity. The above studies fail to provide quantitative support for their conclusions concerning CO2 as relevant buffering agent. More likely, the constant neutralization time after successive alkali exposure may be related to the destruction of the ‘‘skin barrier’’ and unlimited penetration of the applied alkali as suggested by others [29, 30].
Free Amino Acids Free amino acids in the water-soluble portion of the epidermis seem to play a significant role in the neutralization of alkalis within the first 5 min of experimentation [27, 36, 37]. Piper [36] found a good buffering capacity of skin between pH 4 to pH 8 with an optimum at 6.5 well corresponding to the pKa of AA. This observation further indicates that lactic acid may be less relevant in the buffering capacity of skin. Despite the general agreement about the role of amino acids in the neutralization of alkalis, which amino acids are the key buffering agents remains an open question. The AA composition of the upper stratum corneum was reported by Spier and Pascher [33]. Spier and Pascher reported that the free AA account for 40% of the water-soluble substances extracted from the stratum corneum removed by tape stripping [34, 40]. Of the amino acids present the composition was as follows: 20–32% serine, 9–16% citrulline, 6–10% aspartic acid, glycine, threonine, and alanine, and 0.5–2% glutamic acid.
The water-soluble, free AA on the skin surface may originate from five possible sources. 1. Eccrine sweat Sweat contains 0.05% amino acids which remain on the surface of the skin after evaporation. The specific AA found in sweat was not investigated. 2. Degradation of skin proteins Degradation of skin proteins including proteins constituting the desmosomes may be a source for AA such as serine, glycine, and alanine. 3. Hair follicles Citrulline is recognized as a constituent of protein synthesized in the inner root sheath and medulla cells of the hair follicle. Specific proteases release citrulline. Citrulline is also found in proteins in the membrane of the corneocytes [41]. 4. Keratin hydrosylates Although as discussed above the AA composition of Keratin does not correspond with the composition of free AA found the stratum corneum [34, 41, 42]. 5. Keratohyalin granule histidine-rich protein The pool of free amino acids, urocanic acid and pyrrolidone carboxylic acid in mammalian stratum corneum has been shown to be derived principally or totally from the histidine-rich protein of the keratohyalin granules. The time course of appearance of free amino acids and breakdown of the histidine-rich protein are similar, as are the analyses of the free amino acids and the histidine-rich protein. Quantitative studies show that between 70% and 100% of the total stratum corneum-free amino acids are derived from the histidine-rich protein [46, 47]. These results strongly suggest that the free amino acids and/or their metabolites of the stratum corneum might be the final products of a degradation of the histidine-rich protein. Further research needs to be completed in order to identify which of these AAs contribute to the buffering capacity of skin.
Specific Physiologic and Structural Changes in the Stratum Corneum of Elderly Skin: Impact on the Buffering Capacity of Elderly Skin Lipid Content/Sebum Production The brick and mortar model is often used to describe the stratum corneum’s protein-rich corneocytes embedded in a matrix of ceramides, cholesterol, and fatty
Buffering Capacity Considerations in the Elderly
acids, and smaller amounts of cholesterol sulfates, gucosylceramides, and phospholipids. As stated earlier, these lipids form multi-lamellar sheets amidst the intracellular spaces of the stratum corneum critical to the stratum corneum’s mechanical and cohesive properties, enabling it to function as an effective water barrier [18, 48]. Many authors agree that the overall lipid content of human skin decreases with age [48–50], although the proportion of different lipid classes seems to remain fairly constant [7]. Sebaceous gland function is decreased in association with concomitant decrease in endogenous androgen production [51]. This is the likely cause of decreased surface lipid levels in the elderly. In males, sebum levels remain essentially unchanged until the age of 80 years. In women, there is a gradual decrease in sebaceous secretion from menopause through the seventh decade, after which no appreciable change occurs [52]. As discussed in the previous section the lipid layer is presumed to slow down the exposure of any topical insult. Therefore with decreased amount of lipid, any topical insult will more easily overwhelm the buffering system of the stratum corneum.
Water In young skin, most of the water is bound to proteins and appropriately termed bound water [53]. Bound water is important for the structure and mechanical properties of many proteins and their mutual interactions. Water molecules that are not bound to proteins bind to each other and are called tetrahedron or bulk water [53]. In aged skin water is mostly found in the tetrahedron form, bound to itself rather than to other molecules [54]. The lack of interaction between water and surrounding molecules in aged skin leads to variation in the water-soluble portion of the stratum corneum and likely contributes to decreased buffering capacity found in elderly skin. In addition, this chemical change in the water explains why although aged stratum corneum has higher total water content than younger skin it is often dry and weathered [7].
Proteins The majority of proteins in young skin are in helical conformation. This is in contrast to aged skin which can show markedly altered protein conformation such as increased protein folding resulting in less exposure to aliphatic residues to water [53, 54]. Increased protein folding and
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decreased interaction of proteins with water effects the concentration of AA in the stratum corneum which as discussed previously, likely plays an important role in the buffering capacity of skin. In addition the AA composition of proteins and free amino acids in aged skin also differ significantly from that of young skin. There is an increase in the overall hydrophobicity of amino acid in the elderly [55, 56]. As free amino acids are believed to play a key role in stratum corneum buffering capacity this shift in composition, combined with evidence of altered protein tertiary protein structure, provides insight into the diminished buffering capacity in aged individuals. In addition, it should also be noted that the increase in pH of aged skin will also change the fraction of AA in the stratum corneum that are associated or disassociated. Free AA work best as a buffer at pH that is equal to their pKa (i.e., the pH at which 50% of the AA associated and 50% disassociated). Because of the increased baseline pH found in elderly skin the percentage of associated to disassociated AA changes, hence changing the effectiveness of the buffer.
Eccrine Sweat Glands With aging the number of active eccrine sweat glands is reduced and sweat output per gland is diminished in both in rate and amount. Morphologically, the secretory cells flatten and become atrophic. A progressive accumulation of lipofuscin is found in the cytoplasm of the glandular epithelium [51, 57]. Therefore any contribution of eccrine sweat to the buffering capacity would be decreased in aged skin due to the decreased output of sweat overall.
Conclusion Skin’s exquisite buffering capacity has been widely studied in vitro and in vivo, yet further research is required to better understand the exact mechanisms responsible for the buffering capacity of skin. Experimentation reviewed here suggests that AAs are primarily responsible for the neutralization capacity of skin. The exact sources of the amino acids as well as the types of AA that are primarily responsible for the neutralization capacity remain still rather speculative. In addition, it seems that a sweat component increases the neutralization capacity of the epidermis. Whether the buffering component of sweat is additional AA or lactic acid remains unknown.
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While additional components of the epidermis such as sebum, keratin, and CO2 seem not to significantly participate as buffering agents of the epidermis, they still may play a role in the protection of skin from the harm of acids and bases. Sebum may slow down the initial penetration of applied substances. Keratin is important for the hydration of the skin and may contribute to the free AA pool responsible for buffering of applied acids/alkalis. Finally, CO2 may play a role in the buffering capacity of certain compounds under certain circumstances such as after prolonged or repetitive exposure to an alkali. Elderly skin has an increased pH and decreased buffering capacity. These two changes in the physiochemical nature of elderly skin arguably contribute to the fragility of elderly skin by influencing barrier homeostasis, skin integrity/cohesion, susceptibility to infection, and skin sensitivity to topical acids and alkalis. After thorough review of studies investigating the buffering capacity of skin and studies investigating the endogenous mechanisms for restoring and maintaining skin pH, it is interesting that the two topics have been investigated separately without looking for a commonality. It would not be surprising if the mechanisms responsible for maintaining skin pH influence the processes responsible for maintaining skin buffering capacity. The above rationale may shed light on clinical correlation of increased pH and decreased buffering capacity that is seen in certain skin disease [58] and in elderly skin [7]. This theory is supported by the discovery that 70–100% of AAs of the stratum corneum are derived from the degradation of histidine-rich protein in keratohyalin granules which is also one of the essential pathways involved in maintaining skin pH [3, 46, 47]. This theory is further supported by the fact that decreasing skin pH in the elderly via acidic topical products has lead to an increased buffering capacity and reduced skin sensitivity. One study in particular used a preparation acidified with citric acid/ammonium citrate buffer and demonstrated a significant shortening of the alkaline neutralization time in aged skin from 5.3 0.6 to 4.9 0.5 min after 4 weeks application [59]. While more research needs to be conducted on the benefit of topical acidic therapy for aged individuals, this application seems reasonable as many authors have demonstrated the use of acidic topic products or washes on patients with increased pH to help restore integrity/cohesion [22] and barrier recovery [22]. The rich experimental literature, even if old at times, leads the way to utilizing several contemporary methods to further refine insights into skin’s buffering capacity and aging. This capacity, when fully understood, may lead not
only to the potential for decreasing threat of exogenous acids and bases to aged skin, but also for establishing an experimental bases for optimal pH in many cosmetic, pharmacologic, metabolic and toxicologic situations in elderly individuals.
References 1. Heuss E. Die Reaktion des Scheisses beim gesunden Menschen. Monatsh. Prakt. Dermatol. 1892;14:343. 2. Schade H, Marchionini A. Zur physikalischen Cheme der Hautoberflache. Arch Dermatol Syphil. 1928;154:690. 3. Kim M, Patel R, Shinn A. Evaluation of gender difference in skin type and pH. J Dermatol Sci. 2006;41:153–156. 4. Greener B, Hughes A, Bannister N, Douglas J. Proteases and pH in chronic wounds. J Wound Care. Feb 2005;14(2):59–61. 5. Hachem J, Crumrine D, Fluhr J, Brown B, Feingold K. Elias P. pH directly regulates epidermal permeability barrier homeostasis, and stratum corneum integrity/cohesion. J Invest Dermatol. 2003;121: 345–353. 6. Agache P. Measurement of skin surface acidity. In: Agache P, Humbert P, Maibach H (eds) Measuring Skin. Springer, 2004, pp. 84–86. 7. Waller JM, Maibach HI. Age and skin structure and function, a quantitative approach (II): protein, glycosaminoglycan, water, and lipid content and structure. Skin Res Technol. 2006 Aug;12 (3):145–154. 8. Raab W. Skin cleansing in health and idsease. Wien Med Wschr. 1990;141(108):4–10. 9. Segger D, Abmus U, Brock M, Erasmy J, Finkel P, Fitzner A, Heuss H, Kortemeier U, Munke S, Rheinlander T, et al. IFSCC Magazine. 2007;10(2):107–110. 10. Burckhardt W. Beitrage zur Ekzemfrage. II. Die rolle des alkali in Pathogenese des ekzems speziell des Gewerbeekzems. Arch f Dermat U Syph. 1935;173:155–167. 11. Burckhardt W. Beitrage zur Ekzemfrage. III. Die rolle des alkalischadigung der haut bei der experimentellen Sensibilisierung gengen Nickel. Arch f Dermat U Syph. 1935;173:262–266. 12. Burckhardt W. Neure untersuchungen uber die Alkaliempfindlicjkeit der haut. Dermatologica. 1947;94:73–96. 13. Burckhardt W, Baumle W. Die Beziehungen der saurempfindlichkeit zur Alkaliempfindlicjkeit der haut. Dermatologica. 1951;102:294–300. 14. Fore-Pfliger J. The epidermal skin barrier: implications for the wound care practitioner, part I. Adv Skin Wound Care. 2004;17(8): 417–425. 15. Zlotogorski A. Distribution of skin surface pH on forehead and cheek of adults. Arch Dermatol Res. 1987;279:398–401. 16. Thune P, Neilsen T, Hnastad IK, et al. The water barrier function of skin in relation to water content of the stratum corneum, pH and skin lipids. Acta Derm Venerol. 1988;68:277–283. 17. Laufer A, Dikstein S. Objective measurement and self-assessment of skin care treatments. Cosmet Toiletires. 1996;111:91–98. 18. Choi EH, Man MQ, Xu P, Xin S, Liu Z, Crumrine DA, Jiang YJ, Fluhr JW, Feingold KR, Elias PM, Mauro TM. Stratum corneum acidification is impaired in moderately aged human and murine skin. J Invest Dermatol. 2007;127(12):2847–2856.
Buffering Capacity Considerations in the Elderly 19. Hachem JP, Crumrine D, Fluhr J, Brown BE, Feingold KR, Elias P. pH directly regulates epidermal permeability barrier homeostasis, and stratum corneum integrity/cohesion. J Invest Dermatol. 2003;121:345–353. 20. Fluhr JW, Kao J, Jain M, Ahn SK, Feingold KR, Elias PM. Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity. J Invest Dermatol. 2001;117:44–51. 21. Ekholm E, Egelrud T. Expression of stratum corneum chymotrypic enzyme in relation to other markers of epidermal differentiation in skin explant model. Exp Dermatol. 2000;9:65–70. 22. Leveque JL, Corcuff P, de Rigal J, Agache P. In vivo studies of the evolution of physical properties of the human skin with age. Int J Dermatol. 1984 Jun;23(5):322–9. 23. Dunner M. Der Einfluss des Hauttalges auf die Alkaliabwehr der Haut. Dermatologica. 1950;101:17–28. 24. Fishberg E, Bierman W. Acid base balance in sweat. J Biol Chem. 1932;97:433–441. 25. Vermeer D. The effect of sebum on the neutralization of alkali. Dederl Tijdschr V Geneesk. 1950;94:1530–1531. 26. McKenna B. The composition of the surface skin fat (Sebum) from the human forearm. J Invest Dermatol. 1950;15:33–37. 27. Vermeer D, Jong J, Lenestra J. The significance of amino acids for the neutralization by the skin. Dermatologica. 1951;103:1–18. 28. Lincke H. Beitrage zur Chemie und Biologie des Hautoberflachenfetts. Arch f Dermat U Syph. 1949;188:453–481. 29. Neuhaus H. Fettehalt und Alkalineutralisationskahigkeit der haut unter Awendung alkalifrier waschmittel. Arch f Dermat U Syph. 1950;190:57–66. 30. Vermeer D, Jong J, Lenestra J. The significance of amino acids for the neutralization by the skin. Dermatologica. 1951;103:1–18. 31. Schmidt P. Uber die Beeinflussung der Wasserstoffionenkonzentration der Hautoberflache durch Sauren. Betrachtungen uber die Funktionen des ‘‘Sauremantels’’. Arch f Dermat U Syph. 1941;182:102–26. 32. Vermeer D. Method for determining neutralization of alkali by skin. Quoted in Yearbook: Dermat & Syph. 1951;415. 33. Wohnlich H. Zur Kohlehydratsynthase der Haut. Arch f Derm Syph. 1948;187:53–60. 34. Spier H, Pascher G. Quantitative Untersuchungen uber die freien aminosauren der hautoberflache. – Zur frage Ihrer Genese. Klinische Wochenchrift. 1953:997–1000. 35. Sharlit H, and Sheer M. The hydrogen ion concentration of the surface on healthy intact skin. Arch Dermat Syph. 1923;7: 592–598. 36. Piper H. Das Neutralisationsvermogen der haut gegenuber Laugen und seine Beziehung zur Kohlensauteabgabe. Arch f Dermat U Syph. 1943;183:591–647. 37. Jacobi O. Uber die Reaktiosfagigkeit und das Neutralisationsvermogen der lebenden menschlichen Haut. Dermat Wchnschr. 1942;115: 733–741. 38. Lustig B, Perutz A. Ube rein einfaches Verfahren zur Bestimmung der Wasserstoffionenkonzentration der normalen menschlichen Hautoberflache. Arch f Dermat U Syph. 1930;162:129–134. 39. Steinhardt J, Zaiser E. Combination of wool protein with cations and hydroxyl ions. J Biol Chem. 1950;183:789–802.
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40. Green M, Behrendt H. Patterns of Skin pH from Birth to Adolescence with a Synopsis on Skin Growth. Springfield: Charles C Thomas, 1971, pp. 93–100. 41. Peterson LL, Wuepper KD. Epidermal and hair follicle transglutaminases and crosslinking in skin. Mol Cell Biochem. 1984;58 (1–2):99–111. 42. Steinhert P, Freedberg I. Molecular and Cellular biology of Keratins. In: Goldsmith L (eds) Physiology and Molecular Biology of the Skin, 2nd ed. Oxford University Press, 1991, pp. 113–14732. 43. Arnold D. The self disinfecting power of skin. Am J Hyg. 1934;19:217–228. 44. Szakall A. Uber die physiologie der obersten Hautschichten und ihre Bedeutung fur die Alkaliresistenz. Arbeitsphysiol. 1941;11:436–452. 45. Szakall A. Die Veranderungen der obersten Hautschichten durch den Dauergebrauch einiger Handwaschmittel. Arbeitsphysiol. 1943;13:49–56. 46. Scott IR, Harding CR, Barrett JG. Histidine-rich protein of the keratohyalin granules. Source of the free amino acids, urocanic acid and pyrrolidone carboxylic acid in the stratum corneum. Biochim Biophys Acta. 1982;719(1):110–117. 47. Horii I, Kawasaki K, Koyama J, Nakayama Y, Nakajima K, Okazaki K, Seiji M. Histidine-rich protein as a possible origin of free amino acids of stratum corneum. Curr Probl Dermatol. 1983;11:301–315. 48. Rogers J, Harding C, mayo A, Banks J. Rawlings A. Stratum corneum lipids: the effects of ageing and the seasons. Arch Dermatol Res. 1996;288:765–770. 49. Roskos KV. The effect of skin aging on the percutaneous penetration of chemicals through human skin. Dissertation, UCSF, CA. 50. Saint Leger D, Francois AM, Leveque JL, Stoudemayer TJ, Grove GL, Kligman AM. Age-associated changes in the stratum corneum lipids and their relation to dryness. Dermatologica. 1988;177:159–164. 51. Pollack SV. The aging skin. J Fla Med Assoc. 1985;72(4):245–248. 52. Pochi PE, Strauss JS, Downing DT. Age-related changes in sebaceous gland activity. J Invest Dermatol. 1979;73:108–111. 53. Gniadecka M, Nielsen OF, Christensen DH, Wulf HC. Structure of water, proteins, and lipids in intact human skin hair nail. J Invest Dermatol. 1998;110:393–398. 54. Gniadecka M, Nielsen OF, Wessel S, Heidenheim M, Christensen DH, Wulf HC. Water and protein structure in photoaged and chronically skin. J Invest Dermatol. 1998;11:1129–1133. 55. Jacobson T, Yuksel Y, Geesin JC, Gordon JS, Lane AT, Gracy RW. Effects of aging and xerosis on the amino acid composition of human skin. J Invest Dermatol. 1990;95:296–300. 56. Jacobson TM, Umit Yuksel K, Geesin JC, et al. Effects of aging and xerosis on the amino acid composition of human skin. J Invest Dermatol. 1990;95:296–300. 57. Selmanowitz VJ, et al. Aging of the skin and its appendages. In Finch C, Hayflick (eds) Handbook of the Biology of Aging. New York: van Nostrand Reinhol Company, 1977, pp. 496–509. 58. Kurabayahi H, Tamura K, Machida I, Kubota K. Inhibiting bacteria and skin pH in hemiplegia: effects of washing hands with acidic mineral water. Am J Phys Med Rehabil. 2002;81:40–46. 59. Meigel W, Sepehrmanesh M. Untersuchung der pflegenden wirkung und der vertraglichkeit einer cre`me/loti bei alteren patienten mit trockenem hautzustand. Dtsch Derm. 1994;42:1235–1241.
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30 Cellular Energy Metabolism and Oxidative Stress Regina Hourigan
Introduction The objective of this chapter is to provide an overview of how changes to the skin’s energy metabolism systems lead to a decline in function and hence contribute to skin aging. This chapter first discusses how the skin uses energy to maintain its appearance followed by a background on energy production in cells. Defects in energy production are part of the mitochondrial theory of aging, which will be introduced next. Skin-specific examples which support this theory of aging will be given as well as evidence that questions this theory. The examples are divided into chronological skin aging and extrinsic skin aging. Lastly, examples of anti-aging therapies which improve or maintain metabolic functions of the skin are given. This chapter intends to also provide areas for discussion or debate, as there is a cyclic nature to the role of mitochondria in aging. It is not clear if aging causes mitochondrial defects or if mitochondrial defects cause aging. Similarly, while oxidative stress can cause mitochondrial defects, mitochondrial defects can also generate oxidative stress. It is in fact this cyclic nature which may progressively lead to more damage.
Energy Metabolism and the Role of Mitochondria in the Skin Skin is in essence a sacrificial protective coating of the body. It is continually shed to maintain an effective barrier to outside insults. As such, the skin is composed of many types of proliferating cells which have a high energy demand. For example, suprabasal layers of the epidermis and cells within the root of the hair follicle have high metabolic activity associated with the synthesis of keratin and the cornified envelopes. Adenosine triphosphate (ATP) is a vital source of energy for these metabolic activities of skin cells. It is required for proliferation as a result of mitogenic stimuli, collagen synthesis, and DNA repair. It supports functions that maintain skin turnover and the extracellular matrix. A decline or dysfunction in ATP
production impacts the skin’s functions, and ultimately its appearance. Mitochondria can also have an impact on the skin through keratinocyte differentiation. In cell cultures, mitochondria-mediated cell death can trigger keratinocyte differentiation. Characteristics of differentiation (flattened morphology, stratification, and keratin 10 expression) are detected after an reactive oxygen species (ROS)-induced release of cytochrome c and apoptosisinducing factor (AIF) [1].
Background on Mitochondria Structure and Function This section briefly describes the mitochondrial mechanisms for energy production. This provides a brief background for the later discussions. Mitochondria generate ATP, which is used as chemical energy for most eukaryotic cells. They also control cell functions related to cell death, differentiation, and cell signaling. They contain their own genetic code, similar to the nucleus of the cell. Their genetic material is referred to as mitochondrial DNA (mtDNA). The mitochondria are composed of several compartments, enclosed by an inner and outer membrane. The outer membrane contains a protein called porin, which forms aqueous channels allowing for protein transport through the membrane. The inner membrane contains an important mechanism for energy production, the electron transport chain (ETC). The ETC is a major source of ATP production in the cell. It is a series of enzymatic complexes, called complexes I, II, III, IV, and V. It uses molecules derived from fuel sources to produce oxygen. The starting molecules are electron donators, reduced nicotinamide adenine dinucleotide (NADH), and reduced flavin adenine dinucleotide (FADH2). The NADH enters at complex I and FADH2 enters at complex II. At the end of complex IV the output is oxygen, which combines with the electrons and protons to form water. Electron transfer through complexes I–IV is managed by the complexes and electron carriers, such as
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coenzyme Q10 (CoQ10) and cytochrome c. The flow of electrons and the ATP synthesis by OxPHOS is continuous within tissues containing mitochondria [2]. The exchange of electrons from a high-energy state to a lower-energy state generates an electrochemical proton gradient. This gradient provides the energy to drive the phosphorylation of adenosine diphosphate (ADP) to ATP. This process, in which ADP is converted to ATP using inorganic phosphate, is called oxidative phosphorylation (OxPHOS). It occurs in complex V. The ATP is then available for use as chemical energy for the cell [2].
ROS and Mitochondria The majority of electrons travel successfully to successive enzymatic complexes, but some electrons can leak from the ETC. These electrons readily react with the available oxygen to form the ROS superoxide. It is thought that about 1–3% of the O2 reduced in the mitochondria may form superoxide [2]. The mitochondria contain antioxidant defenses to control the level of ROS. Superoxide is converted to hydrogen peroxide and water by the antioxidant enzyme superoxide dismutase. The hydrogen peroxide is converted to water and molecular oxygen by catalase or glutathione peroxidase. Although at times detrimental, ROS is also used for cell signaling [2], for example, for apoptosis of the cell [3]. Damage to the mitochondria increases ROS, which triggers the release of cytochrome c and apoptosis-inducing factor (AIF). Their release initiates the caspase-dependent and caspase-independent cell death pathways to remove the damaged cell [3]. ROS leakage from the ETC increases unchecked ROS, which directly damage the mitochondria. Specific damage will be discussed in the later sections. The damaged mitochondria decline in function, which in turn generates more ROS. This is known as the ‘‘vicious cycle’’ where ROS are both a cause and a consequence of mtDNA mutations. The ‘‘vicious cycle’’ creates an amplifying feedback loop which sustains the damaging effects, even with a small amount of initial insult. The ‘‘vicious cycle’’ is believed to create ongoing elevated levels of stress, such as those found with aging [4, 5]. The ROS and its resulting ‘‘vicious cycle’’ are the foundations of the mitochondrial theory of aging.
Mitochondrial Theory of Aging The free radical theory of aging (FRTA) was formed by Harman, in 1956 [6]. The FRTA proposes that the
underlying source of aging is the accumulation of oxidative damage in macromolecules and tissues. Later, Harman proposed that the mitochondria’s production of the superoxide may be central to the FRTA [7]. This is called the mitochondrial theory of aging. A fundamental part of this theory is that mtDNA is at particular risk for ROS damage. This is proposed because of the proximity of mtDNA to the ROS-producing mitochondrial matrix. The mitochondrial theory of aging is supported by evidence of age-related ROS accumulation and mitochondrial changes. There is an increase in ROS produced from the ETC with aging [8, 9]. Complexes I, II, and III are considered to be the sites of excessive ROS [10–13]. As tissues age, there is lower flux through the ETC and reduced ATP production [14]. The lower flux causes more free electrons to be lost and form ROS [15, 16]. The increased ROS with age can directly damage the structures of the mitochondria itself, such as proteins, lipids, and mtDNA [17–19]. ROS damages mtDNA by creating strand breaks within the mtDNA. Studies of the respiration-dependent mitochondrial processes conclude that mtDNA damage is related to a decline in respiratory processes. These processes include mitochondrial protein synthesis, oxygen consumption, and ATP generation. For example, as the amount of mtDNA damage increases, the mitochondria membrane potential lowers and cannot be maintained. Maintaining membrane potential is critical to the electrochemical proton gradient and OxPHOS. In parallel, cytochrome c is released into the cytoplasm, which activates caspases leading to premature apoptosis [20]. The compromised respiratory processes elevate ROS levels further, creating the above-mentioned ‘‘vicious cycle.’’ It has been debated whether the accumulation of such oxidative damage is a cause or a consequence of aging. In 2007, Muller et al. reviewed the topic of whether oxidative stress determines life span. They conclude that the case for oxidative stress as a life span determinant may be tentatively made for Drosophila melanogaster, but is not certain in humans or mice [21]. Kujoth et al. have proposed accumulation of mtDNA mutations, which promote that apoptosis may be a mechanism driving mammalian aging [22]. Recent work by Doonan et al. found that there is no impact on basal life span with increased levels of superoxide dismutase (through gene manipulation of Caenorhabditis elegans) [23]. While basal life span was not changed, improving organisms’ ability to cope with elevated oxidative stress can lengthen life span. Supplying C. elegans with antioxidant mimetics extended their life span, and normalized prematurely aged organisms’ life spans, during exposure to oxidative stress [24].
Cellular Energy Metabolism and Oxidative Stress
Coping with stresses is critical to survival and longevity. It should be noted that other factors related to coping with stresses, such as inflammation or repair mechanisms are also critical determinants of longevity. In the skin, stressful environments can clearly cause oxidative damage that leads to extrinsic aging and there is likely a place for the mitochondrial theory of aging.
Skin Energy Metabolism and Chronological Aging Skin is aged through two mechanisms: chronological aging (a function of the passage of time) and extrinsic aging (a function of external stress, i.e., photoaging from ultraviolet radiation [UVR]). This section discusses the chronological aging of skin, related to energy metabolism, and will be followed by a separate discussion on extrinsic skin aging and energy metabolism. One approach to studying chronological aging is harvesting the skin cells from variously aged donors and comparing their functions. When skin cells are collected in this manner, there are differences in the metabolic functions of age groups. According to Greco et al., in 2003, human dermal fibroblasts from 51 donors aged 1–103 years showed a clear reduction in mitochondrial processes with age. These included mitochondrial protein synthesis, respiration rate, and coupling of respiration to ATP production. In individuals above 40 years there was a significant decline in the mitochondrial protein synthesis. There was also a significant decrease in endogenous native respiration rate within the age range of 40–90 years. Human skin fibroblasts also had a significant agedependent decrease in the efficiency of respiration and phosphorylation. The ratio of skin cell’s rate of respiration in the presence of ADP to that in the absence of ADP (RCR) is a measure of the OxPHOS’s control of respiration efficiency. This ratio significantly decreased with the age of the donor [25]. In the skin cells, particularly from donors over the age of 40, metabolic functions decline as a function of age. The loss of function of donated cells may have been due to multiple causes experienced over their life span. Another study looked at the targeted influence of older mtDNA on cell functions. In this study, mitochondria from fibroblasts of 21 individuals between the ages of 20 weeks and 103 years were inserted into human mtDNA-less cells. An age-dependent decrease in growth rate and a decline in respiratory rate were detected in the cells receiving the older mitochondria [26]. This observation supports that chronologically aged mitochondria
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can cause characteristics of aging, such as reduced cell growth and respiratory functions. The amount of available energy metabolites in the skin may be thought to be related to the age of the skin, that is, younger skin contains more energetic materials – yet that is not the case observed in vivo. There are no differences in the skin’s basal levels of energy metabolites with age [27]. Using 31P nuclear magnetic resonance spectroscopy, young and old skin (ventral aspect of the wrist) from panelists did not show differences in baseline levels of phosphocreatine, inorganic phosphate, adenosine triphosphate, phosphomono, and phosphodiesters. However, what is significantly different is how the age groups respond to stress. After single exposure to a low, sub-erythema level of UVA irradiation, there were significant differences in the response and recovery of energy metabolism. The older skin showed slower response and recovery than younger skin [27]. This is an indication of the importance of evaluating skin aging characteristics in basal as well as stressed conditions. This is an important consideration when evaluating therapies related to energy metabolism. An anti-aging material may not influence the basal characteristics of the skin but, may be influential in reducing stresses or up-regulating repair, thereby preventing extrinsic aging. The mitochondria and its functions play both causative and effective roles in cell dysfunctions and senescence. Senescent human cell cultures are a model for chronological aging [28]. The loss of mitochondrial functions, as passage number increases, can cause premature senescence in skin cells. It induces a senescent phenotype likely with the increase in ROS. This has been demonstrated by a reduction in the level of OxPHOS in fibroblasts causing a reduction in cell proliferation and premature senescence in human fibroblasts [29]. On one hand, the mitochondria affect senescence but, on the other, changes that occur with senescence can effect mitochondrial respiration. With increasing passage number, senescent fibroblasts show a loss of membrane potential [30] and a decline in ATP production [31]. This may be due to inefficient removal of mitochondrial damage in the cells as proteasomes activity declines. Proteasome inhibition is emerging as a common factor, based on in vitro and in vivo experiments, in aging and age-related diseases. Proteasomes are part of the protein removal system for most eukaryotic cells. They contain proteases, which degrade damaged or unnecessary proteins from the cell. Proteasome activity declines with age in the human epidermis [32]. Keratinocytes that undergo replicative senescence are known to have a reduction in proteasome levels [33]. While oxidative
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damage has been known to cause proteasome dysfunction during aging, Torres and Perez have recently shown that proteasome inhibition is a mediator of oxidative stress and ROS production by affecting mitochondrial function. They proposed that a progressive decrease in proteasome function during aging can promote mitochondrial damage and ROS accumulation [34].
Skin Energy Metabolism and Extrinsic Aging The change in skin appearance from external insults is called extrinsic aging. These insults can include UVR, pollution, and smoking. The major cause of extrinsic aging is UVR, referred to as photoaging. Photoaged skin has many well-accepted characteristics, such as loss of elasticity, reduced hydration, greater barrier damage, to mention a few. The UVR induces theses changes by interacting with and damaging skin structures; i.e., the proteins, DNA, and lipids of the skin. mtDNA can also be damaged by UVR. Greater accumulations of mtDNA damage are found in sun-exposed skin compared to protected skin [35–42]. It is thought that the strand breaks in mtDNA caused by UVR are mediated by ROS, particularly superoxide. The 4,977-base pair (bp) deletion and 3,895-bp deletion mtDNA are among the recognized mtDNA deletions related to skin photoaging [37, 40, 41, 43]. The 4,977-bp deletion is called the common deletion because it is the most prevalent marker of mtDNA damage in humans and is found in several types of tissues. The 3,895-bp deletion, which is less reported, may also play a role in skin photoaging [41]. The 4,977-bp deletion occurs in both in vitro and in vivo skin studies and relates to UVR exposure. The common deletion was found in human skin fibroblasts treated with a repeated, sublethal dose of UVA [36]. Studies with keratinocytes are less numerous, but have also indicated a link between UVR and mitochondrial damage. A single dose of UVB exposure to kertatinocytes induced two mtDNA deletions: the 4,977-bp and a novel 5,128-bp deletion [43]. In vivo, the common deletion can be induced in the dermal tissue of living skin after repeated exposure to UVA radiation. UVA induced a 40% increase in the common deletion in the dermis, but not in the epidermis of the irradiated skin [37]. There are differing findings on the presence of the 4,977-bp deletion in chronologically aged skin. There is evidence for an age-related increase in the 4,977-bp type of deletion in skin mtDNA. The 4,977-bp deletion was not
found in skin samples from donors under the age of 60 years. The frequency of this deletion in skin did increase with age for individuals who were 60–90 years old [37]. Keratinocytes-induced 4,977-bp deletion has, in other cases, not corresponded to the chronological age of the donors (30–78 years). Koch et al. noted slower cell proliferating body sites, that is, blood, brain, and skeletal tissues, and showed correlation between chronological age and increasing mtDNA deletion [40]. The 3,895-bp deletion corresponds to broadspectrum UV exposure in both in vitro and in vivo studies. HaCaT cells (line derived from keratinocytes) exposed to repeated UVA/UVB doses were found to have the mtDNA 3,895-bp deletion [41]. Among skin samples from 42 skin donors, the 3,895-bp deletion was found at a higher amount in ‘‘usually’’ sun-exposed body sites (face, ears, neck, and scalp) compared with ‘‘occasionally’’ exposed sites (shoulders, back, and chest). The deletion was not detected in the body sites that were ‘‘rarely’’ exposed to sunlight. The 3,895-bp deletion induction was in both the epidermis and the dermis of the exposed sites. In the usually exposed sites, the level was almost equal in the epidermis and the dermis. Therefore, mitochondrial damage may serve as a biomarker for cumulative sun exposure [41]. In summary, UV-induced ROS can cause mtDNA damage, which serves as a marker of skin damage. These genetic changes also lead to a decline in mitochondrial function (i.e., ATP production). The UVA-induced common deletion in human dermal fibroblasts corresponds to decreases in oxygen consumption, mitochondrial membrane potential, and ATP content [44]. This leads to compromised mitochondrial respiration. Once compromised in this manner, the mitochondrial respiration causes an increase in ROS produced by the ETC. The intracellular mitochondrial oxidative stress generated under these conditions upregulates matrix metalloproteinase-1 (MMP-1) [44]. MMP-1 is an accepted indicator of aged and damaged skin. MMP-1 is responsible for collagen degradation in photoaged skin [45], the skin of tobacco smokers [46], and chronologically aged (sun-protected) skin [47]. Skin fibroblast studies have shown that in the absence of UVA, a deletion of 4,977-bp causes an increase of MMP-1. UVA-induced common deletion also corresponds to an increase in the expression of MMP-1 without an increase of the tissuespecific MMP inhibitors [44]. The cascade of UV stress, ROS generation, mtDNA damage, and elevated ROS from compromised respiration leads to collagen degradation, a direct factor in the appearance of aged skin. While this cascade has a linear sequence of events, there is also a feedback loop (the ‘‘vicious cycle’’
Cellular Energy Metabolism and Oxidative Stress
mentioned earlier) created by the ROS. The ROS that cause mitochondrial respiratory dysfunction lead to more ROS being produced by the cell. There has been recent evidence of the ‘‘vicious cycle’’ in the skin as measured by sustained mtDNA damage. The common deletion was found to remain in UV-exposed skin for up to 16 months after experimental treatment (in vivo). In some cases, the deletion continued to increase after UV exposure has ceased [37]. This increase after exposure and sustained level of mtDNA deletion may be due to the ongoing cycle of ROS generation and mtDNA mutations. This ‘‘vicious cycle’’ may be a source of chronic oxidative stress within the skin. The chronic stress may be a factor in premature aging as the ROS may then interact with skin proteins, initiate inflammation, and promote extracellular matrix degradation (> Fig. 30.1). While ultraviolet radiation has been well studied for its impact on skin photoaging, recent studies are now showing an emerging role for infrared (IR) radiation in photoaging.
. Figure 30.1 The potential vicious cycle of mitochondrial damage and skin aging
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IR accounts for more than half of the solar energy that reaches the human skin [48]. It is divided into IRA, IRB, and IRC. IRB and IRC do not penetrate deeply into the skin, while more than 65% of IRA reaches the dermis [48]. Near IRA (760–1,440 nm) can induce MMP-1 expression in ex vivo fibroblast cells [49]. The production of ROS from IRA stress originates from the mitochondrial ETC. Cultured fibroblasts treated with antioxidants are protected from the IRA radiation and do not upregulate MMP-1 expression [49]. In vivo studies also show that skin responds to IRA radiation with upregulation of MMP-1 in the dermis [50]. In parallel, there is a decrease in the skin’s antioxidant content [50]. In vivo, the skin was protected from IRA-induced MMP-1 upregulation with the use of antioxidants [50]. The implication is that the mitochondria ROS signaling that leads to MMP-1 collagen degradation can be prevented by antioxidants.
Select Anti-aging Therapies This section highlights select cellular respiration anti-aging approaches. As mentioned above, one protective approach is with antioxidants. DNA, lipids, and proteins are known to be protected by the application of antioxidants on the skin. Antioxidants can regulate the transfer of electrons or quench the free radicals escaping from the ETC. This can mitigate the effects of photoaging through the prevention of oxidative damage and the related damage to mitochondrial functions. Some examples of antioxidants are glutathione, CoQ10, and N-acetyl cysteine (NAC). At low concentrations of the antioxidant glutathione, UVB-induced mtDNA deletions have been prevented [43], giving further evidence that mtDNA damage by UVR is mediated by ROS. At higher levels of glutathione, when it acts as a reductive antioxidant (electron donor) and hence a deleterious agent, the protective effect ceases and the mtDNA deletions return [43]. CoQ10 is a known antioxidant found in the mitochondria, and serves to carry electrons in the ETC. Its level in the skin declines with age and UV stress [51]. A series of in vitro and in vivo experiments by Hoppe et al. have shown the benefits of CoQ10 in prevention of skin aging. Topical application of CoQ10 reduced wrinkle depth and level of oxidation in vivo. CoQ10 is also effective in protecting the DNA of keratinocytes from UVA-induced oxidative stress. CoQ10 is also able to reduce the expression of collagenase in dermal fibroblasts following UVA irradiation [52]. N-acetyl cysteine (NAC) is an antioxidant which increases the intracellular concentration of glutathione
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(GSH) [53]. Lipoic acid and NAC supplementation of Alzheimer’s patient’s fibroblasts protected the mitochondria from oxidative stress in vitro [54]. Although not performed with skin cells, NAC oral supplementation of mice increases the mitochondrial respiration of senescent liver cells. The liver cells’ ETC complexes had higher activity, while levels of protein carbonyls, a marker of protein oxidation, were reduced [55]. Another therapy for the improvement of skin’s energy metabolism is to provide the skin with energy supplementation. One approach is with the amino acid creatine. Creatine is the precursor to phosphocreatine. Phosphocreatine is synthesized in the mitochondria by creatine kinases. Phosphocreatine can donate a phosphate group to ADP to produce ATP. This provides an additional reserve of ATP that can be used by cells for metabolic activity. Creatine does not offer antioxidant or UV protection, rather its protective effect is from increasing cell energy reserves. Lenz et al. observed the photoprotective effect of creatine on human skin cells in vitro and in vivo [56]. Supplementation of normal human fibroblasts with creatine during repeated UVA exposure showed a mitigation of mtDNA mutations as well as the normalization in oxygen consumption and MMP-1 production [44]. Creatine also prevents the common deletion, and inhibitors of creatine block these effects [44]. These data show that while UVA reduces mitochondrial function, supplementation with creatine can mitigate these effects. The researchers suggest that the prevention of UVA-induced common deletion may be from creatine’s ability to normalize the cell’s energy status. This prevents an upregulation of a deleterious respiratory chain, which generates more ROS [44]. Supplementation with energy precursors also allows for more efficient repair. Maes et al. have shown, in a skin model, that DNA repair from UV stress exposure is enhanced with creatine [57]. In human clinicals, with a formulation containing creatine, acetyl-L-carnitine, and NADH reduced the appearance of aging [58]. The researchers believed that the enhanced repair was due to the increased availability of ATP that the creatine provided. Under the stress of UV, the cells can synthesize the needed repair enzymes using this additional ATP [57, 58]. Combinations of the above therapies are also effective. In vivo, a combination of CoQ10 and a stabilized form of creatine in a topical emulsion improved signs of skin aging, including density of the dermal papillae [59]. Protecting cellular energy metabolism of skin can improve protection from UV stress, provide energy for repair systems during stress, and cause positive changes to the skin morphology.
Conclusion Aging is a complex topic involving all of the functions of the skin, and its underlying mechanism is difficult to attribute to any single biological source. Multiple studies give evidence that mitochondrial damage is either the cause or a marker of age-related dysfunctions in the skin. The damage, and related decline in mitochondrial functions, can create aged skin appearance. The contribution of mitochondrial damage to skin aging may be amplified by the presence of a vicious cycle. Mitigation and prevention of this mitochondrial damage alleviates the signs of skin aging. While by no means the only factor in skin aging, alterations to the skin’s energy metabolism systems lead to a decline in function and hence contribute to skin aging.
Cross-references > Alterations
of Energy Metabolism in Cutaneous Aging
References 1. Tamiji S, et al. Induction of apoptosis-like mitochondrial impairment triggers antioxidant and Bcl-2-dependent keratinocyte differentiation. J Invest Dermatol. 2005;125:647–658. 2. Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine, 4th edn. New York: Oxford University Press, 2007. 3. Danial NN, Korsmeyer SJ. Cell death: critical control points. Cell. 2004;116:205–219. 4. Linnane A, et al. Mitochondrial DNA mutations as an important contributor to ageing and degenerative diseases. Lancet. 1989; 1:642–645. 5. Bandy B, Davison AJ. Mitochondrial mutations may increase oxidative stress: implications for carcinogenesis and aging? Free Radic Biol Med. 1990;8:523–539. 6. Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol. 1956;11(3):298–300. 7. Harman D. The biologic clock: the mitochondria? J Am Geriatr Soc. 1972;20(4):145–147. 8. Barja G. Mitochondrial free radical production and aging in mammals and birds. Ann N Y Acad Sci. 1998;854:224–238. 9. Moghaddas S, Hoppel C, Lesnefsky EJ. Aging defect at the QO site of complex III augments oxyradical production in rat heart interfibrillar mitochondria. Arch Biochem Biophys. 2003;414:59–66. 10. Chen Q, et al. Production of reactive oxygen species by mitochondria central role of complex III. J Biol Chem. 2003;278(38): 36027–36031. 11. Turrens JF. Superoxide production by the mitochondrial respiratory chain. Biosci Rep. 1997;17(1):3–8. 12. Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol. 2003;552(2):335–344.
Cellular Energy Metabolism and Oxidative Stress 13. Barja G. Mitochondrial oxygen radical generation and leak: sites of production in states 4 and 3, organ specificity, and relation to aging and longevity. J Bioenerg Biomembr. 1999;31(4):347–366. 14. Harper, et al. Age-related increase in mitochondrial proton leak and decrease in ATP turnover reactions in mouse hepatocytes. Am J Physiol Endocrinol Metab. 1998;275:197–206. 15. Qian T, Nieminen AL, Herman B, Lemasters JJ, Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes. Am J Physiol. 1997;273:C1783–C1792. 16. Chen Q, Lesnefsky EJ. Depletion of cardiolipin and cytochrome c during ischemia increases hydrogen peroxide production from the electron transport chain. Free Radic Biol Med. 2006;40:976–982. 17. Chen JJ, Yu BP. Alterations in mitochondrial membrane fluidity by lipid peroxidation products. Free Radic Biol Med. 1994;17: 411–418. 18. Sohal RS, Dubey A. Mitochondrial oxidative damage, hydrogen peroxide release, and aging. Free Radic Biol Med. 1994;16:621–626. 19. Agarwal S, Sohal RS. DNA oxidative damage and life expectancy in houseflies. Proc Natl Acad Sci USA. 1994;91:12332–12335. 20. Mandavilli BS, Santos JH, Van Houten B. Mitochondrial DNA repair and aging. Mutat Res. 2002;509:127–151. 21. Muller F, et al. Trends in oxidative aging theories. Free Radic Biol Med. 2007;43(4):477–503. 22. Kujoth GC, et al. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science. 2005;309:481–484. 23. Doonan R, et al. Against the oxidative damage theory of aging: superoxide dismutases protect against oxidative stress but have little or no effect on life span in Caenorhabditis elegans. Genes Dev. 2008;22:3236–3241. 24. Melov S, et al. Extension of life-span with superoxide dismutase/ catalase mimetics. Science. 2000;289(5484):1567–1569. 25. Greco M, et al. Marked aging-related decline in efficiency of oxidative phosphorylation in human skin fibroblasts. FASEB J. 2003;17: 1706–1708. 26. Laderman KA, et al. Aging-dependent functional alterations of mitochondrial DNA (mtDNA) from human fibroblasts transferred into mtDNA-less cells. J Biol Chem. 1996;271:15891–15897. 27. Declercq L, et al. Age-dependent response of energy metabolism of human skin to UVA exposure: an in vivo study by 31P nuclear magnetic resonance spectroscopy. Skin Res Technol. 2002;8:125–132. 28. Cristofalo VJ, et al. Use of the fibroblast model in the study of cellular senescence. In: Barnett Y, Barnett C (eds) Aging Methods and Protocols. Totowa: Humana Press, 2000, pp. 26. 29. Stockl P, et al. Sustained inhibition of oxidative phosphorylation impairs cell proliferation and induces premature senescence in human fibroblasts. Exp Gerontol. 2006;41:674–682. 30. Mammone T, Gan D, Foyouzi-Youss R. Apoptotic cell death increases with senescence in normal human dermal fibroblast cultures. Cell Biol Int. 2006;30:903–909. 31. Zwerschke W, et al. Metabolic analysis of senescent human fibroblasts reveals a role for AMP in cellular senescence. Biochem J. 2003;376(Pt 2):403–411. 32. Bulteau AL, Petropoulos I, Friguet B. Age-related alterations of proteasome structure and function in aging epidermis. Exp Gerontol. 2000;35:767–777. 33. Petropoulos I, et al. Increase of oxidatively modified protein is associated with a decrease of proteasome activity and content in aging epidermal cells. J Gerontol Biol Sci Med Sci. 2000;55:B220–B227. 34. Torres CA, Perez VI. Proteasome modulates mitochondrial function during cellular senescence. Free Radic Biol Med. 2008;44: 403–414.
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35. Yang JH, Lee HC, Lin J, Wei YH. A specific 4977-bp deletion of mitochondrial DNA in human ageing skin. Arch Dermatol Res. 1994;286:386–390. 36. Berneburg M, et al. Singlet oxygen mediates the UVA-induced generation of the photoaging-associated mitochondrial common deletion. J Biol Chem. 1999;274(22):15345–15349. 37. Berneburg M, et al. Induction of the photoaging-associated mitochondrial common deletion in vivo in normal human skin. J Invest Dermatol. 2004;122(5):1277–1283. 38. Birch-Machin MA, et al. Mitochondrial DNA deletions in human skin reflect photo-rather than chronologic aging. J Invest Dermatol. 1998;111(4):709–710. 39. Ray A, et al. The spectrum of mitochondrial DNA deletions is a ubiquitous marker of ultraviolet radiation exposure in human skin. J Invest Dermatol. 2000;115:674–679. 40. Koch H, Wittern K-P, Bergemann J. In human keratinocytes the common deletion reflects donor variabilities rather than chronologic aging and can be induced by ultraviolet a irradiation. J Invest Dermatol. 2001;117:892–897. 41. Krishnan K, Harbottle A, Birch-Machin MA. The use of a 3895 bp mitochondrial DNA deletion as a marker for sunlight exposure in human skin. J Invest Dermatol. 2004;123:1020–1024. 42. Eshaghian A, et al. Mitochondrial DNA deletions serve as biomarkers of aging in the skin, but are typically absent in nonmelanoma skin cancers. J Invest Dermatol. 2006;126:336–344. 43. Ji F, et al. Novel mitochondrial deletions in human epithelial cells irradiated with an FS20 ultraviolet light source in vitro. J Photochem Photobiol. 2006;184(3):340–346. 44. Berneburg M, et al. Creatine supplementation normalizes mutagenesis of mitochondrial DNA as well as functional consequences. J Invest Dermatol. 2005;125:213–220. 45. Brennan M, et al. Matrix metalloproteinase-1 is the major collagenolytic enzyme responsible for collagen damage in UV-irradiated human skin. Photochem Photobiol. 2003;78(1):43–48. 46. Lahmann C, et al. Matrix metalloproteinase-1 and skin ageing in smokers. Lancet. 2001;357(9260):935–936. 47. Varani J, et al. Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin. J Invest Dermatol. 2000;114:480–486. 48. Schroeder P, Haendeler J, Krutmann J. The role of near infrared radiation in photoaging of the skin. Exp Gerontol. 2008;43:629–632. 49. Schroeder P, et al. Cellular response to infrared radiation involves retrograde mitochondrial signaling. Free Radic Biol Med. 2007;43(1):128–135. 50. Schroeder P, et al. Infrared radiation-induced matrix metalloproteinase in human skin: implications for protection. J Invest Dermatol. 2008;128:2491–2497. 51. Podda M, et al. UV radiation depletes antioxidants and causes oxidative damage in a model of human skin. Free Radic Biol Med. 1998;24:55–65. 52. Hoppe U, et al. Coenzyme Q10, a cutaneous antioxidant and energizer. BioFactors. 1999;9(2–4):371–378. 53. Zafarullah, et al. Molecular mechanisms of N-acetylcysteine actions. Cell Mol Life Sci. 2003;60(1):6–20. 54. Moreira P, et al. Lipoic acid and N-acetyl cysteine decrease mitochondrial-related oxidative stress in Alzheimer disease patient fibroblasts. J Alzheimer’s Dis. 2007;12(2):195–206. 55. Miquel J, et al. N-Acetylcysteine protects against age-related decline of oxidative phosphorylation in liver mitochondria. Eur J Pharmacol. 1995;292:333–335.
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Transdermals, Dermatologicals, and Cosmetic Actives, 1st edn. Ames: Wiley-Blackwell, 2006, pp. 117–124. 59. Blatt T, et al. Stimulation of skin’s energy metabolism provides multiple benefits for mature skin. The Fourth Conference of the International Coenzyme Q10 Association. BioFactors Special Issue 2005;25(1–4):179–185.
23 Changes in Nail in the Aged Nelly Rubeiz . Ossama Abbas . Abdul Ghani Kibbi
Introduction Nail complaints are very common among the elderly. These may represent either normal age-related nail alterations or nail dystrophies that tend to be more common in the elderly secondary to several factors including impaired circulation, faulty biomechanics, infections, neoplasms, and dermatological or systemic diseases [1, 2]. Alone or in combination, these factors may affect the matrix, nail bed, hyponychium, or nail folds leading to secondary abnormalities in the nail plate. These nail alterations may be symptomatic and may impair daily activities or may be associated with significant cosmetic problems leading to a negative psychological impact. Knowledge of these age-related nail changes and dystrophies as well as their underlying causes is important in order to effectively reach an accurate diagnosis and thus provide better care for the nail concerns of this large and growing elderly population.
Normal Senile Nail Changes The age-associated nail changes include characteristic changes in morphology, growth, chemical composition, and histology of the nail unit [1, 2]. The mechanisms underlying these changes are not clear, but may be due to a dysfunctional circulation at the distal extremities or to the ultraviolet radiation effects.
Age-Related Morphological Nail Changes These include changes in color, contour, surface, and thickness of the nail plate [1, 2]. Among the most common nail color changes observed in elderly people is a yellow to gray discoloration with dull, pale, or opaque appearance. The normally smooth texture of the nail plate tends to become increasingly friable with advancing age leading to splitting, fissuring, and longitudinal superficial or deep striations [1, 2]. In general, nail plates are thicker in men than women, where the normal average thickness of fingernails and toenails is 0.6 and 1.65 mm in men
and 0.5 and 1.38 mm in women, respectively. With advancing age, the nail plate thickness may become thicker, thinner, or remain the same [1, 2]. ‘‘Neapolitan nails’’ is a peculiar discoloration observed in up to 20% of people above 70 years and manifests as three horizontal bands of white (proximal), pink (middle), and opaque (distally) discoloration in addition to an absent lunula [1]. One study found that this peculiar nail alteration is significantly associated with osteoporosis and thin skin, and suggested an abnormality in collagen leading to these changes in nail bed, bone, and skin [3]. Although usually seen in liver cirrhosis and chronic congestive heart failure, Terry’s nails, a type of apparent leukonychia characterized by a distal transverse pink band and proximal white band, is occasionally seen as a part of the normal aging process [4]. Senile nail contour changes include an increase in the transverse convexity and a decrease in the longitudinal curvature [1, 2].
Age-Related Nail Growth Rate Changes The normal average growth rate of fingernails and toenails is 3.0 and 1.0 mm/month, respectively. In elderly people, there is a decrease in this rate of growth by approximately 0.5%/year after the age of 25 years [1, 2].
Age-Related Changes in the Chemical Composition of the Nail Plate The nail plate is made up of tightly layered cornified cells that are generated by the nail matrix epithelium and consists mainly of intermediate filamentous proteins or keratins (80–90% which are of hard keratins) [5]. The keratins are embedded in a matrix composed of nonkeratin proteins (high-sulfur and high-glycine/tyrosine proteins). Other nail plate constituents include water (7 and 18%), lipids (0.1–5%), and trace elements (mainly iron, zinc, calcium, and magnesium) [1, 2, 5–7]. Normally, the nail plate calcium (Ca) content is low (0.2% by weight), while the sulfur content is high (10% by weight) [4]. It is believed that the relatively higher sulfur content
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_23, # Springer-Verlag Berlin Heidelberg 2010
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to Ca content contributes to nail plate hardness, especially that the former is a reflection of the cysteine disulfide bonds which stabilize fibrous proteins [6]. Even though the Ca content of the nail does not seem to contribute to the hardness of the nail plate, a recent study revealed that both fingernail and toenail Ca concentration decrease with advancing age while magnesium (Mg) concentration tend to increase [6]. Interestingly, this study also showed that measurement of the Ca and Mg contents of the nail plate may be used as osteoporosis predictor. The iron content usually decreases in senile nails [1, 2]. Another constituent of nail plates are membrane-forming integral lipids such as cholesterol and cholesterol sulfate, which are also present in keratinized skin areas and hair. There is an age-related significant decrease in fingernail cholesterol sulfate levels which has been reported in women and may explain the higher incidence of brittle nails in the elderly category [8].
Age-Related Changes in Nail Histology Compared to young individuals, the dermis of the nail bed usually exhibits elastic tissue degeneration and thickening of the blood vessels. In addition, the keratinocytes of the nail plate are commonly enlarged and show increased remnants of keratinocyte nuclei, which are also known as pertinax bodies [1, 2, 9]. To the best of the authors’ knowledge, there are no reports describing the changes that may occur in the nail matrix as a result of aging.
Age-Related Nail Dystrophies Several nail disorders affect the population at large and may appear with advancing age and include, without order of frequency or age-related prevalence, brittle nails, infections (especially onychomycosis), onychauxis, onychocryptosis, onychoclavus, onychogryphosis, onychophosis, splinter hemorrhages, subungual hematoma, and malignancies of the nail apparatus [1, 2, 7–12].
Brittle Nails Brittle nail disorder is characterized by increased nail plate fragility and is considered to be a polymorphic abnormality affecting about 20% of the population with higher incidence in women and the elderly [7]. Clinically, brittle nails manifest with onychoschizia and onychorrhexis, the severity of either may be variable [7].
Onychoschizia, which results from impairment of intercellular adhesion of nail plate corneocytes, usually presents as a lamellar splitting of the distal portion and free edge of the nail plate, and a transverse splitting secondary to breakage of the lateral edges. Underlying causes are usually exogenous and include trauma, repetitive cycles of wetting and drying, the action of fungal proteolytic products, and the use of chemicals or cosmetics such as nail enamel solvents, cuticle removers, and nail hardeners, among others. Onychorrhexis, which results from abnormalities in epithelial growth and keratinization secondary to nail matrix involvement, usually manifests as longitudinal thickening, splitting or ridging of the nail plate, and/or multiple splits leading to triangular fragments at the free edge. Abnormalities of vascularization and oxygenation (such as arteriosclerosis or anemia), dermatological (inflammatory diseases and disorders of cornification), and systemic diseases (endocrine, metabolic, etc.) are among the various factors that may underlie the abnormalities of growth and keratinization responsible for onychorrhexis. Recently, a composite scoring system assessing the severity of nail brittleness based on the degree of ridging, nail thickness, lamellar, longitudinal, and transverse splitting has been proposed [7]. Management of brittle nails may not be easy or simple [7]. The initial therapeutic approach is to determine the predominance of either onychoschizia or onychorrhexis. Underlying factors should then be identified and if possible, be corrected. After that, general and specific measures may be followed. Nail hydration by 15 min daily soaks of the nail and by the application of emollients rich in phospholipids may be useful. Strengthening the nail plate may be accomplished by the application of nail hardeners containing formaldehyde; however, these should be utilized cautiously because they may lead to brittleness, onycholysis, and subungual hyperkeratosis. Although enamel application may mechanically protect the nail and fill the fractures; its removal however, may lead to substantial dehydration. Several studies have shown that daily oral intake of biotin (2.5 mg/d) for 1.5–15 months may be beneficial; however, these studies are small and not double blind placebo-controlled [7, 10, 11].
Infections Infections by different pathogens may affect the nail plate either primarily or through extension from involved adjacent structures such as the nail folds [1, 2, 10–12].
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Onychomycosis, a fungal infection of the fingernails and/or toenails, is the most common infection and represents up to half of all nail diseases [12]. It affects 10–20% of adults, especially the elderly. Multiple factors are associated with an increased risk of onychomycosis including old age, male gender, underlying medical diseases (diabetes, peripheral arterial disease, and immunodeficiency), smoking, and predisposing genetic factors [12]. More than 90% of onychomycosis cases are caused by dermatophytes, among which Trichophyton rubrum and T. mentagrophytes are the most common. Other less commonly encountered causative organisms include yeasts such as Candida and nondermatophyte molds such as Scopulariopsis brevicaulis and Scytalidium hyalinum [12]. Five clinical subtypes of onychomycosis are recognized [12]. Distal and lateral subungual onychomycosis (DLSO), the most common subtype usually caused by T. rubrum, manifests as subungual hyperkeratosis, onycholysis, nail thickening, and discoloration secondary to fungal invasion which starts at the hyponychium and spreads proximally along the nail bed. Superficial onychomycosis usually presents as white (caused by T. mentagrophytes) or black (caused by dematiaceous fungi) patchy nail discoloration due to fungal invasion of the dorsal surface of the nail plate. Proximal subungual onychomycosis (PSO) commonly affects immunocompromised individuals and presents clinically as a white spot under the lunula that progresses distally. It results from fungal invasion (usually T. rubrum), from the proximal nail fold to the nail plate. Endonyx onychomycosis (EO) is an uncommon form caused by T. soudanense; it resembles DLSO; however, the nail thickness is within normal and there is no hyperkeratosis or onycholysis. Total dystrophic onychomycosis (TDO) is an advanced form, characterized by progressive nail plate destruction leaving an exposed abnormally thickened nail bed. TDO may be observed in immunodeficient patients such as those with chronic mucocutaneous candidiasis and is fairly acute or may be progressive representing an end stage of other forms of onychomycosis. Effective treatment of onychomycosis entails making an accurate diagnosis and identifying the causative pathogen [12, 13]. Several diagnostic methods including KOH-based microscopy, fungal cultures, and histopathology with PAS may be used alone or in combination; the latter being the most sensitive [12, 13]. The treatment options include oral and/or topical antifungal agents, mechanical or chemical treatments, or a combination of these. The choice of the therapy should be individualized based on several factors such as the causative agent, the potential for drug interactions and side effects, the
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number of nails involved, the severity of onychomycosis, and the cost. Currently, terbinafine appears to be the most effective oral agent for treating dermatophyte onychomycosis, especially in elderly patients due to its fungicidal action, safety, and low potential for drug interaction [13]. The azoles (ketoconazole, fluconazole, itraconazole) can also be used but are generally considered to be less effective than terbinafine as they are fungistatic rather than fungicidal [13]. Paronychia, seen occasionally in elderly patients, is an acute or chronic infection of the nail folds which may lead to secondary changes in the nail plate [1, 2]. Acute paronychia, usually caused by Staphylococcus aureus, most commonly presents as tender erythematous swelling of only one nail and is typically trauma-induced. Management includes abscess drainage, warm saline soaks, and the use of topical or systemic antibiotics. Chronic paronychia is commonly caused by Candida species or Gramnegative bacteria and presents clinically as red and swollen nail folds with loss of cuticle and multiple secondary transverse ridges in the nail plate. Keeping the nail fold dry coupled with topical antifungal or antiseptic agents are the treatment of choice. Elderly patients, similar to infants and immunosuppressed hosts, are prone to uncommon presentations of Sarcoptes scabiei infestation in which all skin surfaces such as the scalp and face, as well as nails may be affected. The mite may inhabit and persist in subungual hyperkeratotic debris, leading to prolonged infestations and/or epidemics among elderly patients and those caring for them in nursing homes. Cutting the nails as much as possible and brushing their tips with a scabicide is an adjunct modality to the antiscabetic treatment [1, 2].
Onychauxis Onychauxis is a localized hypertrophy of the nail plate which presents clinically as discoloration, hyperkeratosis, loss of nail plate translucency, and often subungual hyperkeratosis [1, 2]. The underlying cause/s may be related to the aging process or to faulty biomechanics that tend to be more common in the geriatric population. Overlapping and underlapping toes, digiti flexi (contracted toes secondary to buckling of toes induced by shortening of the controlling muscles), or foot-to-shoe incompatibility are examples of these faulty mechanics. Onychauxis may be associated with pain, and, with time, may be complicated by distal onycholysis, increased risk for onychomycosis, subungual hemorrhage, and/or subungual ulceration.
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Partial or total debridement of the thickened nail plate should be done periodically. Other treatment options include the use of electric drills, 40% or higher urea paste, or nail avulsion. In complicated cases or those with recurrences, chemical or surgical matricectomy may be done to achieve permanent ablation of the affected plate.
Onychoclavus Onychoclavus, also known as subungual corn, is a hyperkeratotic process most commonly located under the distal nail margins [1, 2]. It results from chronic minor trauma and persistent localized pressure by bony abnormalities such as digiti flexi, rotated fifth toes, foot-to-shoe incompatibility, or hallux valgus (the great toe rotates toward the second toe). It usually presents as a tender dark area under the nail plate (most commonly the great toenail) and may be confused with benign and malignant subungual melanocytic lesions and subungual exostosis [1, 2]. The treatment of this condition is surgical removal of the hyperkeratotic tissue and the correction of the underlying bony abnormality.
Onychogryphosis Onychogryphosis, also known as ram’s-horn nail, is a term used to describe thickening and enlargement of the nail plate, most commonly the great toenails [1, 2]. The affected nail plate is usually brownish to opaque in color, grows faster on one side than the other, may have many grooves and transverse striations, and is commonly associated with nail bed hypertrophy. This nail dystrophy is common among the elderly population, and if untreated, may lead to a walking disability. In patients with diabetes mellitus or peripheral vascular disease, onychogryphosis may be complicated by subungual gangrene due to pressure effects [1]. Infrequent nail cutting, trauma, foot-to-shoe incompatibility, and bony abnormalities such as hallux valgus are responsible for its pathogenesis. Onychogryphosis should be distinguished from hemionychogryphosis, which is characterized by the lateral growth of the nail plate from the onset as a complication of persistent congenital malalignment of the great toenails. Beyond cosmetic considerations, treatment of onychogryphosis in the elderly may be mandatory in order to prevent disability and its complications [1, 2]. Conservative management with the use of an electric drill or burr
to file the involved nail plate is the initial step followed by removal of subungual hyperkeratosis and subsequent periodic nail plate trimming. Other more radical approaches such as nail avulsion, with or without matricectomy may be valuable in selected patients.
Onychophosis Onychophosis is a localized or diffuse hyperkeratosis under the nail plate (subungual), on the lateral or proximal nail folds, or in the space between the nail plate and nail folds [1, 2]. The first and the fifth toes are the digits of predilection. Multiple nail and adjacent soft tissue abnormalities including onychocryptosis, nail fold hypertrophy, and onychomycosis may be the underlying causes of onychophosis. Other external causes such as repeated minor trauma and foot-to-shoe incompatibility may be contributing. Several treatment modalities may be used to treat onychophosis and include keratolytic agents (urea 20% or salicylic acid 6–20%), nail packing, and, if needed, surgical excision. Recurrences may be prevented by wearing appropriate comfortable shoes to minimize pressure effects of the nail plate on surrounding nail folds.
Onychocryptosis (Ingrown Toe Nail) Although more commonly observed in young adults, onychocryptosis may occasionally be encountered in the elderly causing significant pain, difficulty walking, and disability [1, 2, 14]. It occurs when the lateral nail plate penetrates the adjacent nail fold as a result of nail plate over-curvature, subcutaneous in-growing toenail, and/or lateral nail fold hypertrophy. Clinically, patients commonly present with tenderness and inflammation of the lateral nail fold, which at times, may be associated with granulation tissue formation and secondary infection. The most common underlying causative factor/s include improper nail cutting, ill-fitting or high-heeled shoes, hyperhidrosis, long toes, prominent nail folds, and bony abnormalities such as hallux valgus. The management of ingrown toenail consists of treating the acute signs and symptoms and correcting the underlying predisposing factors [1, 2]. There is a evidence that the best chance for complete cure is to excise the lateral nail plate, to curette the granulation tissue, and to perform lateral matricectomy [1, 2, 14]. This procedure may be complicated by postoperative nail bed infection
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or by regrowth of a nail spicule secondary to incomplete matricectomy. In addition this procedure, not uncommonly, may lead to recurrences and poor cosmetic results. Recently, surgical decompression of the ingrown toenail without matricectomy has been proven to be very effective. In this approach, a large volume of soft tissue around the nail plate is removed and the inflammation is relieved [14]. The advantage of this maneuver is complete preservation of the nail anatomy and function with excellent therapeutic and cosmetic results.
Splinter Haemorrhages Splinter hemorrhages are linear discolorations under the nail plate that progress over a period of few days from an initial red color to a dark brown or black color [1, 2]. The location of the splinter hemorrhages may give leads to the underlying pathogenesis. Splinter hemorrhages located in the middle or distal third of the nail plate are usually associated with trauma, while those located proximally are commonly associated with systemic diseases such as infective endocarditis, cholesterol emboli, or connective tissue disorders. Proximal type-splinter hemorrhages especially those associated with systemic diseases are generally more common among young adults, whereas several studies have shown that trauma-associated distal splinter hemorrhages are observed frequently in the elderly population. Trauma-induced splinter hemorrhages commonly resolve on their own, while the proximal-type splinter hemorrhages require treatment of the underlying systemic disorder.
Subungual Hematomas Subungual hematomas are common among the elderly and are most frequently induced by trauma, which may or may not be remembered [1, 2]. The event may result in nail bed laceration and bleeding under the nail plate. Amyloidosis, diabetes mellitus, or anticoagulant therapy may also be less common causes of sunbungual hematomas. Early on, it presents as a tender red subungual discoloration that tends to move forward and becomes bluish and less painful with time. The forward and distal movement of this discoloration under the nail plate is a clinical clue that serves to distinguish this lesion from melanocytic proliferations including melanoma. Occasionally, distal onycholysis with subsequent spontaneous nail plate avulsion may occur.
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Reassurance and observation of the nail is the main management strategy. However, in acute painful cases, pressure may be relieved by drilling a hole through the nail plate. Chronic cases are best left to heal spontaneously after ruling out melanoma.
Bowen’s Disease of the Nail Apparatus Bowen’s disease of the nail unit, also known as in situ epidermoid carcinoma, is a nonaggressive malignancy most commonly originating in the epithelium of the nail folds or grooves [15]. The incidence is highest in patients aged between 50 and 69 years, and usually affects the fingers, particularly the thumb [15]. Classical presentations include subungual or periungual ulcerated hyperkeratotic or papillomatous proliferations with associated onycholysis. Rarely, this condition may present as LM or erythronychia. Ulceration or bleeding is usually indicative of invasion which may be deep and reach the underlying contiguous bone in less than 20% of patients. Distant metastatic rate is usually low. The etiology remains unclear; trauma, X-ray exposure, arsenic, chronic paronychia, and human papilloma virus (HPV) infection have all been implicated. The latter has been implicated because HPV 16, 34, and 35 have been detected in many cases of in situ and invasive Bowen’s disease of the nail apparatus, and this raised speculation about a role for genital-digital transmission of the virus. Mohs’ micrographic surgery is the treatment of choice for this condition [15]. Other less effective modalities have been used and include regular excision, electrosurgery, liquid nitrogen, imiquimod, photodynamic therapy, intra-arterial infusion with methotrexate, and radiation therapy. Amputation of the distal phalanx should be done in case of bone involvement. Regular follow-up is essential in view of the potentially polydactylous nature of this disease.
Nail Apparatus Melanoma Nail apparatus melanoma (NAM) usually occurs in Japanese and African Americans, with a relative incidence of 23 and 25%, respectively [16]. It is rarely observed in the white population. It most commonly occurs in the elderly with a mean age at diagnosis of 60–70 years [16]. The most frequent histogenetic type is acral lentiginous melanoma. The classical presentation is a solitary longitudinal melanonychia (LM) of the thumb, index finger, or big toe. In addition, Hutchinson sign (brown or black pigment
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extension from the matrix and nail bed onto the surrounding tissues) may be present and accounts for the radial growth phase of this melanoma. Rarely, a homogeneous or irregular black spot in the matrix or nail bed may be the only sign of a subungual melanoma. Although NAM appears to have a worse prognosis than its cutaneous counterpart, this may be related to the delay in diagnosis of the former. A strong index of suspicion is required when confronted with an isolated LM, especially in an elderly patient [16]. If a biopsy is obtained and the diagnosis is confirmed, treatment of NAM is then tailored based on the stage of the melanoma. Total excision of the entire nail apparatus or Mohs’ surgery is the treatment of choice for in situ melanoma, while invasive melanomas should be managed with distal phalanx amputation. Adjuvant chemotherapy may also be needed in advanced cases.
Other Nail Conditions Several other conditions should be kept in mind when evaluating nail changes in an elderly patient. These include nail changes associated with cutaneous inflammatory disorders (such as psoriasis or lichen planus) [17], nail cosmetics [18, 19], systemic disorders commonly observed in the elderly (such as renal disease) [20], or medications as the elderly patients are usually on multidrug therapy (such as anticoagulants, anticonvulsants, or beta-blockers) [21].
Conclusion Elderly patients may complain of nail changes and dystrophies that may be of cosmetic concern, cause pain, affect daily activities, or be malignant. Awareness of these conditions is essential to reach the correct diagnosis and provide appropriate management.
References 1. Cohen PR, Scher RK. Geriatric nail disorders: diagnosis and treatment. J Am Acad Dermatol. 1992;26(4):521–531.
2. Singh G, Haneef NS, Uday A. Nail changes and disorders among the elderly. Indian J Dermatol Venereol Leprol. 2005;71(6):386–392. 3. Horan MA, Puxty JA, Fox RA. The white nails of old age (neapolitan nails). J Am Geriatr Soc. 1982;30(12):734–737. 4. Saraya T, Ariga M, Kurai D, et al. Terry’s nails as a part of aging. Intern Med. 2008;47(6):567–568. 5. Lynch MH, O’Guin WM, Hardy C, et al. Acidic and basic hair/nail (‘hard’) keratins: their colocalization in the upper cortical and cuticle cells of the human hair follicle and their relationship to ‘soft’ keratins. J Cell Biol. 1986;103:2593–2606. 6. Ohgitani S, Fujita T, Fujii Y, et al. Nail calcium and magnesium content in relation to age and bone mineral density. J Bone Miner Metab. 2005;23(4):318–322. 7. van de Kerkhof PC, Pasch MC, Scher RK, Kerscher M, Gieler U, Haneke E, Fleckman P. Brittle nail syndrome: a pathogenesis-based approach with a proposed grading system. J Am Acad Dermatol. 2005;53(4):644–651. 8. Brosche T, Dressler S, Platt D. Age-associated changes in integral cholesterol and cholesterol sulfate concentrations in human scalp hair and finger nail clippings. Aging (Milano). 2001;13(2):131–138. 9. Lewis BL, Montgomery H. The senile nail. J Invest Dermatol. 1955;24(1):11–18. 10. Colombo VE, Gerber F, Bronhofer M, Floersheim GL. Treatment of brittle fingernails and onychoschizia with biotin: scanning electron microscopy. J Am Acad Dermatol. 1990;23:1127–1132. 11. Hochman LG, Scher RK, Meyerson MS. Brittle nails: response to daily biotin supplementation. Cutis. 1993;51:303–305. 12. Gupta AK, Ricci MJ. Diagnosing onychomycosis. Dermatol Clin. 2006;24(3):365–369. 13. Gupta AK, Tu LQ. Therapies for onychomycosis: a review. Dermatol Clin. 2006;24(3):375–379. 14. Noe¨l B. Surgical treatment of ingrown toenail without matricectomy. Dermatol Surg. 2008;34(1):79–83. 15. Baran R, Richert B. Common nail tumors. Dermatol Clin. 2006; 24(3):297–311. 16. Andre´ J, Lateur N. Pigmented nail disorders. Dermatol Clin. 2006; 24(3):329–339. 17. Holzberg M. Common nail disorders. Dermatol Clin. 2006; 24(3):349–354. 18. Dahdah MJ, Scher RK. Nail diseases related to nail cosmetics. Dermatol Clin. 2006;24(2):233–239. 19. Rich P. Nail cosmetics. Dermatol Clin. 2006;24(3):393–399. 20. Tosti A, Iorizzo M, Piraccini BM, et al. The nail in systemic diseases. Dermatol Clin. 2006;24(3):341–347. 21. Piraccini BM, Iorizzo M, Starace M, et al. Drug-induced nail diseases. Dermatol Clin. 2006;24(3):387–391.
34 Climacteric Aging and Oral Hormone Replacement Therapy Pascale Quatresooz . Claudine Pie´rard-Franchimont . Ge´rald E. Pie´rard
Introduction In affluent societies of the West, a woman’s appearance is largely appreciated through her skin aspect, which is thought to reflect in part her general health. Inevitably, skin like any other human tissue, undergoes regressive changes with age. Menopause is the time when permanent cessation of menstruation occurs following the loss of ovarian activity. The prefix ‘‘meno,’’ meaning month, is derived from the Greek, and it has been used to refer to the menstrual cycle. ‘‘Pause’’ indicates the cessation of the process. The transition from regular ovulatory cycles to the menopausal state is not an instantaneous event. Rather, a series of progressive hormonal and clinical alterations reflects the decline in the ovarian activity. The period of time between the reproductive period of life and the postmenopausal years is referred to as perimenopause or climacteric. It includes the last years prior to menopause when endocrinologic, clinical, and biologic changes associated with menopause are occurring, as well as the first year following menopause. Postmenopause, on the other hand, is defined as the year of menopausal amenorrhea and the time thereafter. Aging of humans is a physiologic process characterized by a progressive loss in homeostatic capacity of the organism, ultimately increasing the vulnerability to environmental threats and to certain disease status. Obviously, the aging process evolves at different rates among individuals of the same age. In addition, any given subject shows a variable senescence status among his/her organs, and among each of the constituent tissues, cells, and subcellular structures. In addition, intracellular and extracellular molecules are also involved differently in aging. Within each organ system, aging usually manifests as a progressive and almost linear reduction in maximal function and reserve capacity. Menopause is probably an exception to this timetable as it appears as a major turning point of importance in women’s life. In addition, the menopause effects on skin are intermingled with age-associated physiological decrements resulting from acute and chronic
environmental insults. Menopause appears to spot a decline in skin qualities [1]. Any part of the skin is subject to alterations, including the epidermis, dermis, hypodermis, and hair. Life expectancy of women is substantially longer than that of men, but women often experience greater burdens of morbidity and disability. In many societies, the rapid trend in the aging population, combined with the increasing feminization of aging, contributes to the need for a sharp focus on gender issues. As the proportion of older women grows at rapid rates in the global population, the challenges of learning more about the skin condition of this group is welcomed. One of these priorities involves discovering more about the physiology and treatment of menopause and the climacteric period. Both risk factors and health needs are likely to change as women enter the climacteric period. Menopause has been shown to have a potential role in the etiology of some age-related diseases and particular physiological conditions. The continuing increase of woman life expectancy has resulted in a marked increase of women who live years beyond menopause. Indeed, women can nowadays expect to live onethird of their lives in a potential hormonally deficient state. Using age 50 as a proxy for menopause, about 25 million women undergo menopause each year. By 2030, the world population of postmenopausal women is expected to increase to 1.2 billion, with 47 million new entrants each year [1].
Menopause in the Overall Aging Process Menopause appears as a milestone in the women aging process, which is a universal and global evolution showing many different characteristics. It is recognized that the multifacet process of aging is different among organisms. Two distinct classifications of life evolution and aging histories are of major importance when considering aging models. The first classification distinguishes species exhibiting a clear distinction or not exhibiting a clear
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_34, # Springer-Verlag Berlin Heidelberg 2010
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distinction between germ cells and somatic cells. The second classification identifies semelparous species, reproducing only once in their lifetime, and iteroparous species, which reproduce repeatedly. The concept of aging is most clearly defined in iteroparous species, which have a distinct soma separate from the germ line. Aging needs to be clearly qualified when applied to species with other kinds of life history. It is mistaken, for example, to regard the postreproductive end of life of semelparous species, which usually occurs in highly determinate fashion, as being comparable with the more protracted process of senescence in iteroparous species. Physical growth and senescence are both characterized by cumulative progression of interlocking biologic events. They are not always separated in the timetable of life as they may proceed in tandem. There is evidence that depletion in estrogens exert a prominent influence on aging of a series of body systems including bones, the cardiovascular system, and the skin [2]. For years, the rationale for hormone replacement therapy (HRT) in menopausal women appeared straightforward for many physicians. Such replenishment therapy convincingly showed evidence for alleviating the skin atrophy and xerosis in postmenopausal women [1–3].
Skin Aging in Perspective Conceptually, global human aging may be perceived as one single basic process of physiological decline progressing with age. Over the past decades, the understanding of aging skin has considerably expanded, with emphasis on differentiating true chronologic aging changes from photoaging resulting from habitual chronic sun exposure [4]. The action spectrum of photodamages is not fully characterized but it is acknowledged that the cumulative effects from repeated exposures to suberythemal doses of ultraviolet B (UVB) and UVA in human skin are involved in these processes. The role of UVB in elastin promoter activation in photoaging is evident. UVA also contribute significantly to long-term actinic damage, and the spectral dependence for cumulative damages does not parallel the erythema spectrum for acute UV injury on human skin. The near infrared radiations bring additional deleterious effects participating in skin aging. Such a concept based on a duality in skin aging has been challenged because it may appear as an oversimplification in the reality of life. Another more diversified classification of skin aging in seven distinct types was offered [5]. The most important variables include the endocrine and overall metabolic status, the past and
present lifestyle, and several environmental threats including cumulative UV and infrared radiations, and repeated mechanical solicitations by muscles and external forces such as earth gravity (> Table 34.1). In this framework, the climacteric aging is individualized and emphasized in the endocrine type of aging. The global aging is considered to result from the cumulative or synergistic effects of each specific cause. Increased awareness of the distinct age-associated physiologic changes in the skin including the menopause effects allows for more effective skin care regimens, preventive measures, and dermatologic treatment strategies. The immutability of skin aging is challenged by this way. In this context, skin aging appears as a notoriously complex process. In particular, the ideal appearance, structural integrity, and functional capacity of the skin require an adequate balance between many hormonal influences. Any alteration in this controlled system results in significant changes in skin qualities [6]. Among hormones, estrogens and the other sex steroids have profound influences on both skin development and composition [1–3, 6, 7]. The relative hypoestrogenemia associated with menopause contributes to, and probably exacerbates any other deleterious effect of age. Therefore, a gender perspective is required for a full understanding of skin aging. Both from the physiological and psychosocial viewpoints, the determinants of global aging are closely related to the skin aspect and to the gender. Yet until recently the specific gender perspective of aging was rather neglected by investigators. However, the past decade or so has witnessed progresses in understanding the hormonal involvement in the global aging process [6].
. Table 34.1 Types of cutaneous aging Aging type Genetic
Determinant factor Genetic (premature aging syndromes, phototype-related)
Chronologic
Time
Actinic
Ultraviolet and infrared irradiations
Behavioral
Diet, tobacco, alcoholic abuse, drug addiction, facial expressions
Endocrinological Pregnancy, physiological, and hormonal influences (ovaries, testes, thyroid) Catabolic
Chronic intercurrent debilitating disease (infections, cancers)
Gravitational
Earth gravity
Climacteric Aging and Oral Hormone Replacement Therapy
Gender-Linked Aging The effects of estrogen have been studied on several body systems including the skin [1–3]. Since its introduction as a therapeutic agent about 60 years ago, estrogen is acknowledged to exhibit anti-aging effects on women’s skin because several critical functions of the skin are hormonedependent. In particular, estrogen receptors and their associated proteins have been identified [1, 7, 8]. The normal ovarian cycle is the result of a complex interaction between the hypothalamus, pituitary gland, and ovaries. It is further modulated by higher cortical centers, the thyroid gland, the adrenals, and some peripheral hormonal production. The ovulatory cycle starts with the recruitment of a number of follicles from which one becomes dominant and is the source of ovulation. From puberty to menopause, about 200,000 follicles give rise to 500 mature oocytes. Hence, atresia appears as the dominant and continuous process in ovarian physiology. This is a key element, which leads to menopause [1]. During the normal menstrual cycle, estradiol is the dominant estrogen, reaching a peak level at the time of ovulation. Circulating levels of FSH and LH are characterized by a mid-cycle surge. During the climacteric period, the function of the ovaries is progressively failing. The transition from regular ovulatory cycles to the perimenopause and menopause is characterized by variations in cycle length and bleeding pattern. Women who experience menopause at a young age usually have a short transition phase. By contrast, menopause of later occurrence is associated with a variety of long and short intermenstrual bleeding episodes, and an overall increased mean cycle length. During the transition phase of perimenopause, there is large variability in sex steroid production including estrogen release. The amount of circulating estradiol varies from cycle to cycle, probably representing varying degrees of follicular maturation and function. The perimenopausal ovary requires greater amounts of FSH to stimulate estrogen production. Contrary to older belief, estradiol levels do not gradually wane in the years before menopause, but remain in the normal range until follicular growth and development cease [1]. In contrast to estrogens and progestins, androgen levels remain stable during this transitional period. Androstenedione, testosterone, dehydroepiandrostenedione (DHEA), and DHEAsulfate (DHEAS) do not show any change in circulating concentrations prior to menopause. The most sensitive measure for declining ovarian function during perimenopause relies on the assessments of serum gonadotropins, particularly FSH. There is a dramatic rise of FSH during menopause, followed by a
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slow decline over the ensuing decades. LH levels may remain normal in the face of elevated FSH.
Skin Climacteric Aging Estrogens and other sex steroids exert profound influences on both skin biology and composition. Thus, it is believed that adequate hormone levels are required to control the skin structural integrity and functional capacity. In addition to estrogen and androgen receptors in the skin, aromatase activity is present in fibroblasts, adipocytes, and sebocytes in postmenopausal women. As a result, androgens are possibly switched in situ to estrogens. Sex steroids clearly exert a key role in the skin aging process as evidenced by the accelerated decline in skin appearance from the perimenopausal years onwards. These changes have not been studied thoroughly, although histological findings have demonstrated that the estrogen and progesterone receptors in the skin show a relative decline in their expression from the time of the climacteric [7]. Estrogen receptors have been identified in cells of both the epidermis and the dermis [1, 8]. However, their regional distribution within the skin varies considerably in keeping with the concentration seen within the female genital tract. A high estrogen to androgen receptor ratio is present in the vagina, and a reverse ratio with an increase in androgen and a decrease in both estrogen and progesterone receptors is present in the vulva. The menopause and its specific HRT still leave a great many challenges unresolved at the level of the skin. In particular, they address the issues of HRT effects on a series of physiological functions of the epidermis and dermis. In that field, dissension and controversy are rife. Glaring discrepancies are present in the current literature. In a global view, HRT appears to markedly improve climacteric changes in many organs including the skin [9–11]. The bulk of recent studies confirms that estrogens or estro-progestins effectively suppress the climacteric syndrome and genital atrophy, while significantly decreasing the risk of osteoporotic fractures. The influence of menopause on skin and its correction by HRT and specific topical treatments may prove to be difficult to objectivate by clinical inspection alone. Several relevant aspects are, however, conveniently rated in a semiquantitative manner. The visual and tactile perception of skin qualities is a valuable tool in clinical dermatocosmetology. However, it lacks sensitivity and reproducibility when comparative evaluations are made over a prolonged period of time. In addition, the external appearance is sometimes misleading compared to the
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actual changes induced by treatments including HRT. By contrast, noninvasive objective methods of biometrology are well suited for improving the reliability and preciseness of assessments. Some skin changes of climacteric aging are detected as early as in the early postmenopausal years, when HRT might be of benefit to control them. Cutaneous changes observed in the decade following menopause are both age- and hormone-related [9, 10, 12]. Postmenopausal women commonly complain of generalized xerotic, easily bruising and wrinkled skin. Dermal thickness apparently decreases with time after menopause. It has been reported that the decline in dermal collagen occurs at a rapid rate immediately after menopause, and becomes more gradual thereafter [1] Approximately 30% of skin collagen was reported to be lost in the first 5 years after menopause, with an average decline of about 2% per postmenopausal year over a period of 20 years [1]. In this context, it is difficult to differentiate the consequences of menopause from age-associated changes related to a decline in growth hormone [6]. Indeed, both estrogen and growth hormone depletions are combined in aging women. The relative estrogen reduction at the perimenopause contributes to and exacerbates the negative effects of age. As a consequence, the effects of HRT on skin have deservedly attracted much interest [1, 12, 13], although the issue remains controversial [12, 14, 15]. Anyway, the bulk of the literature indicates that hypoestrogenemia has a detrimental effect on skin collagen content, which is partially addressed by HRT. The maximum effect at preventing skin aging appears to occur when HRT is initiated early in the perimenopausal years [1]. However, shortterm treatments fail to bring significant improvements in the skin condition [14, 15]. Some controversial data are also found in the literature [12, 16].
HRT and the Dermal Extracellular Matrix The dermis is a tough connective tissue matrix supporting the various structures embedded in it. It contains highly stable fibers, predominantly made of collagen and elastin. Collagen represents about 80% of the dry weight of the adult skin. It exhibits high tensile strength and prevents the skin from being torn by overstretching. Elastic fibers, which compose about 5% of the dermis serve to recoil the skin to its initial shape after deformation. Fibroblasts and dermal dendrocytes synthesize and control all the components of the extracellular matrix. Light microscopy reveals that the collagen network of sun-protected skin areas are thinner and less compact in aged people.
The interstitial material between the collagen bundles contains hyaluronic acid and other glycosaminoglycans. Interest in molecular biology and morphology have deferred comprehension of more important structural changes in the collagen network. It may become considerably distorted by lifelong mechanical stresses. Moreover, the number of dermal cells declines with age and they exhibit the shape of a shrunken fibrocyte, becoming narrower with a much cytoplasm, suggesting a decreased metabolic activity. A number of studies on the HRT effects on the dermis focused on changes in its thickness, collagen content, and mechanical functions. HRT administration modalities were clearly different among trials. Various estrogens were used in combination or not with cyclic administration of progesterone derivatives to prevent endometrial hyperplasia. Most often, the information has been discussed collectively without distinguishing the effects of estrogens from those of estrogens and progesterone derivatives in combination [1]. Globally, the dermal collagen content and the dermal thickness appear to be maintained in HRT receivers compared to age-matched untreated women [1, 9, 10]. In women with a lowered skin collagen content, estrogen replenishment is believed to be initially of corrective and later of prophylactic value, while in those women with mild reduction of collagen content in the early menopausal years estrogens are of prophylactic value only [1, 16, 17]. Thus, a depletion in skin collagen may be in part corrected but not overcorrected. The replenishment in skin collagen content may show some regional variability with a more pronounced effect on the abdomen than thigh [1]. At present, no consensus has been reached about the value of HRT on climacteric aging of the dermis. Some authors deny any significant effect [14, 15]. Others feel that there are different levels of skin response with good and poor responders [11, 17–19]. The latter poor responsive result may correspond to smokers or to women who have only recently entered the menopausal period and have not yet lost estrogen-replaceable collagen [1]. The water content stored in the dermis is bound to the hydrophilic glycosaminoglycans. Such feature helps protecting the skin against excessive tissue compression while maintaining its suppleness. Estrogens increase dermal hygroscopic properties, probably through enhanced synthesis of dermal hyaluronic acid [1]. A specific role for versican, if any, is not firmly established. The quantitative changes and the decrease in compactness of the collagen bundles in the dermal matrix lead to progressive skin slackness. The resulting aging
Climacteric Aging and Oral Hormone Replacement Therapy
aspect is characterized by a progressive increase in extensibility associated with a loss of elasticity [20, 21]. Some wrinkles are the result of these functional changes [11]. The climacteric period appears to be responsible for wrinkles particularly on the forearms and face. Several controlled trials have shown the benefit of HRT for mitigating these changes [17, 18]. Fine wrinkling, atrophy, and a progressive deepening of facial creases ensue. These skin alterations were reported to be partially reversed in postmenopausal women receiving estrogen or combined HRT [18, 22]. The marked increase in skin extensibility commonly occurring in untreated perimenopausal women, appears limited by HRT, which therefore helps prevent skin slackness [18, 22]. Hence, HRT may exert a beneficial effect on the facial skin by reducing the age-related rheological changes without, however, limiting the number and depth of wrinkles [9, 13, 18, 22]. The maximal effect at preventing skin aging appears to occur when HRT is started early [10].
HRT and the Dermal Microvasculature Flushes at menopause appear to be caused by a prominent vasodilatation particularly in the face, neck, chest, palms, and soles. Their prevalence during the early menopausal years is in part explained by the loss of peripheral vascular control seen in association with estrogen deficiency. This phenomenon is corrected by HRT leading to abolition of the flush. Indeed, estrogens appear to enhance both endothelium-dependent and endothelium-independent vasodilatation in the skin of women [23–25]. A quantitative biometrological study showed a higher red intensity value (parameter a*) in menopausal women receiving HRT for at least 1 year [12]. However, the maximum inducible vasodilation in the forearm skin was reported to be reduced in postmenopausal women receiving estrogen replacement and premenopausal women, compared with untreated postmenopausal women [23]. The beneficial effect of HRT on the skin blood flow has, however, been challenged. HRT users may have fewer chronic leg ulcers and pressure-induced ulcers [26]. Estrogen might increase the wound healing rate in the elderly. This finding warrant confirmation before recommending HRT to improve wound healing.
HRT, Epidermis, and Lips Stress-induced premature senescence (SIPS) occurs after many different sublethal stresses such as those induced by H2O2, other oxygen species, and a variety of chemicals.
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Cells in replicative senescence share common features with cells in SIPS including morphology, senescenceassociated b-galactosidase activity, cell cycle regulation, gene expression, and telomere shortening. Telomere shortening is then attributed to the accumulation of DNA single-strand breaks induced by oxidative stress. Thus, SIPS could be a mechanism of the in vivo accumulation of senescent-like cells in the skin, and DNA damage plays a key role in skin aging and photoaging. The estrogen depletion possibly promotes SIPS. Xerosis is an alteration of the stratum corneum known as dry skin to the laity. This condition results from an altered desquamation process that is often associated with decreased hydration of the upper layers of the stratum corneum and with weakening of the barrier function of the skin. The hydration, the water-holding capacity, and the barrier function of the stratum corneum appear to be increased following HRT [12, 27, 28]. Lip properties are quite distinct from those of the face and other body areas. Lip tissues are subjected to repeated mechanical and other physical and chemical stresses. Age-related changes in geometrical dimensions of lips have been reported with resulting alteration of the levels of extensibility and contractibility [29, 30]. There was also marked differences in the hydration level of the surface as the upper lip appeared more hydrated than the lower one. It was claimed that any hormonal effect was most unlikely in age-related changes in lip surface hydration and lip mechanical properties [30].
HRT and Pilosebaceous Follicles Hair loss, particularly the frontal fibrosing alopecia, is apparently associated with menopause, but it is not corrected by HRT. Tibolone, which is an alternative to HRT may increase the severity of diffuse alopecia and induce facial hypertrichosis [31]. Sebum excretion on facial skin shows large interindividual differences. In nonsupplemented menopausal women sebum excretion has been reported to increase in the perimenopause and later on declines with chronologic aging [9, 30, 32]. HRT-treated women show less prominent variations. However, the benefit differs among women and it remains hardly predictable. Globally HRTmight increase the casual sebum level [11], but there is a lack of consensus about that aspect. Sebaceous glands are privileged targets for sex steroids, particularly 5a-dihydroxytestosterone. Other hormonal controls and neuropeptides are also operative on sebocytes. As a result, the sebum production may be regarded as a marker of some specific hormonal changes.
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The sebaceous gland apparatus is an androgen target exhibiting the highest androgen receptor density in human skin. During climacteric aging, one could expect changes in the sebocyte proliferation, intracellular lipid synthesis, sebum transit time in the follicle, storage in the infundibulum reservoir, rheology and capture at the surface, and inside the stratum corneum. The modifications in the balance in sex hormones at the menopause are often believed to initiate the observed changes in sebum physiology. The decline in estrogen combined with a minimal decrease in androgens results in a relative increase in the androgen–estrogen balance. This should theoretically not hinder the sebocyte activity. These hormonal changes can also affect other segments of the sebaceous follicle, in particular, the size of the sebum reservoir. This is indeed evidenced by the progressive enlargement of the follicular openings. Although sex hormones are tentatively offered as the agents responsible for the objective changes, other hormones and nonhormonal aspects of aging cannot be dismissed. There are quite few objective studies evaluating the amount of sebum released at the skin surface. In addition, in many instances the number of subjects was obviously too low and precluded any sound conclusion. Some studies showed that sebum excretion decreased with aging. In particular, sebum excretion in menopausal women appeared lower than in nonmenopausal women [30]. By contrast, it was claimed to increase in menopausal women under HRT [9, 12, 30]. Contrasting data were reported in another controlled study involving large numbers of women [33]. Data showed that the sebum excretion changes in postmenopausal women were more likely related to hormones than to aging [33]. There was a large diversity among individual values of sebum output at the skin surface. In untreated women, a significant decline in sebum excretion rate accompanied by an increase in both the sebum replacement time and the mean pore size were evidenced during the first decade after menopause. The sebum excretion rate and casual level showed a wide range of interindividual differences early after menopause. These physiological changes were less prominent in women receiving HRT. It was concluded that postmenopausal aging affects the sebum production, but HRT does not significantly control the complex process of seborrhoea. However, HRT mitigates the progressive enlargement of the openings of the sebum follicular reservoir. As a consequence of the diversity of hormonal signals to the sebaceous apparatus, sebum excretion varies according to age, gender, pregnancy, and postmenopause. However, at any given age in men and women, the sebum excretion rate differs between individuals over a wide range. In addition, there is a huge overlap between data
gained in both genders. Hence, it is not the amount of circulating androgens but rather the receptivity of the target tissues that accounts for interindividual differences in sebum excretion. It is clear that additional factors are likely to be operative. The effect of the climacteric and postmenopausal age upon the sebaceous gland function has not been thoroughly and adequately studied using recent biometrological methods. It is generally acknowledged that the sebum dynamics varies throughout adult life. In women, it was reported that the sebum production remained almost stable over about 3 decades and dropped significantly in the age range of 50–59 years. However, those views were challenged. HRT appears to reduce moderately the effects of postmenopausal aging on the sebum rheology. In addition, the follicular pores are kept narrower compared to the skin of untreated women. Indeed, estrogens unquestionably suppress human sebaceous secretion at high pharmacological dosages. It is debatable, however, whether they have any significant effect at physiological levels and whether they play any sizeable part in normal control of the gland. It should be stressed that contraceptives show a moderate sebosuppressive activity in acne-prone young women suffering from increased seborrhea. It is possible that HRT has no effect when seborrhoea is absent or discrete. This does not exclude the possibility of an effect in severe cases. Nevertheless, it should be noted that chronological aging by itself likely mitigates seborrhea.
HRT, Skin, and Bones Menopause has been shown to have a potential role in the etiology of some major age-related diseases including osteoporosis. The one area that has fulfilled the hope of the HRT research has been the changes that occur in the skin and bones. The changes occurring in the dermis and bone, both in the climacteric and with HRT apparently parallel each other [3, 34–36]. There is also a correlation between some skin biomechanical properties and bone density [19, 34, 36]. It is probably the combination of skin thickness, dermal biomechanical functions, and bone mineral density that presents the greater sensitivity and specificity in identifying women vulnerable to osteoporotic fractures after menopause.
Conclusion Most women associate the middle years of life with a negative experience. In particular, it is generally
Climacteric Aging and Oral Hormone Replacement Therapy
acknowledged that a series of skin changes occur when women traverse the menopause and the years beyond. A gender perspective is indispensable for a full understanding of sex-hormone-sensitive cells of the skin. Yet until recently, some of these aspects have rather been neglected by biomedical researchers. However, the concept of climacteric and postmenopausal aging affecting the skin has progressively emerged in recent years. It was particularly studied at the level of the tensile strength of the dermis affected by atrophy and wrinkling. Climacteric xerosis is also recognized and most probably represents the consequence of a defect in the process of desquamation. The administration of HRT appears both safe and effective, provided adequate patient selection is made, and contraindications and appropriate use of hormones (nature, dosages, regimens, routes of administration) are respected. HRT increases the well-being as well as some somatic features in menopausal women. It remains that at present, the pros and cons of HRT make it a complex issue for the physicians taking care of skin changes. All the foregoing findings indicate that chronological aging, the climacteric estrogen deficiency, and HRT exert profound effects on various parts of the skin. In many cases the deleterious effects of low estrogenemia on the skin are reflected in the internal organs. It is acknowledged that skin during the climacteric suffers from some decline in its aspect and physical properties. HRT appears to protect in part the skin from some of the negative changes. HRT acts on the skin at several different sites and thus exhibits a multifactorial effect. The effects can be mediated by a direct hormonal effect on cells enriched in the adequate receptors. These stimulated cells can further produce some paracrine signals to other cells, which are thus indirectly influenced by HRT. It is the interplay between the various skin cell types and their signaling pathways that probably control the skin aspect and healthy look. At least the skin represents the one target organ where the HRT benefits are readily visible to the woman and her relatives.
Cross-references > Aging Genital Skin and Hormone Replacement Therapy
Benefits
References 1. Raine-Fenning NJ, Brincat M, Muscat-Baron Y. Skin ageing and menopause: implications for treatment. Am J Clin Dermatol. 2003;4:371–378. 2. Verdier-Sevrain S, Bonte F, Gilchrest BA. Biology of estrogens in skin: implication for skin aging. Exp Dermatol. 2006;15:83–94.
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3. Dunn LB, et al. Does estrogen prevent skin aging ? Results from the First National Health and Nutrition Examination Survey (NHANES I). Arch Dermatol. 1997;133:339–342. 4. Farage MA, et al. Intrinsic and extrinsic factors in skin ageing: a review. Int J Cosmet Sci. 2008;30:87–95. 5. Pie´rard GE. The quandary of climacteric skin ageing. Dermatology. 1996;193:273–274. 6. Quatresooz P, et al. Skin in maturity. The endocrine and neuroendocrine pathways. Int J Cosmet Sci. 2007;29:1–6. 7. Nelson LR, Bulun SE. Estrogen production and action. J Am Acad Dermatol. 2001;45:S116–S124. 8. Slominski A. Neuroendocrine system of the skin. Dermatology. 2005;211:199–208. 9. Callens A, et al. Does hormonal skin aging exist? A study of influence of different hormone therapy regimens on the skin of postmenopausal women using non-invasive measurement techniques. Dermatology. 1996;193:189–291. 10. Sauerbronn AV, et al. The effect of systemic hormonal replacement therapy on the skin of postmenopausal women. Int J Gynaecol Obstet. 2000;68:35–41. 11. Quatresooz P, et al. Skin climacteric aging and hormone replacement therapy. J Cosmet Dermatol. 2006;5:3–8. 12. Guinot C, et al. Effect of hormonal replacement therapy on skin biophysical properties of menopausal women. Skin Res Technol. 2005;11:201–204. 13. Quatresooz P, Pie´rard GE. Downgrading skin climacteric aging by hormone replacement therapy. Exp Rev Dermatol. 2007;2:373–376. 14. Oikarinen A. Systemic estrogens have no conclusive beneficial effect on human skin connective tissue. Acta Obstet Gynecol Scand. 2000;79:250–254. 15. Phillips TJ, et al. Does hormone therapy improve age-related skin changes in postmenopausal women? J Am Acad Dermatol. 2008;59:397–404. 16. Brincat MP, Muscat Baron Y, Galea R. Estrogens and the skin. Climacteric. 2005;8:110–123. 17. Castelo-Branco C, et al. Facial wrinkling in postmenopausal women. Effects of smoking status and hormone replacement therapy. Maturitas. 1998;29:75–86. 18. Pie´rard-Franchimont C, et al. Climacteric skin ageing of the face. A prospective longitudinal intent-to-treat trial on the effect of oral hormone replacement therapy. Maturitas. 1999;32:87–93. 19. Pie´rard GE, Vanderplaetsen S, Pie´rard-Franchimont C. Comparative effect of hormone replacement therapy on bone mass density and skin tensile properties. Maturitas. 2001;40:221–227. 20. Pie´rard GE, et al. Effect of hormone replacement therapy for menopause on the mechanical properties of skin. J Am Geriatr Soc. 1995;43:662–665. 21. Hermanns-Leˆ T, et al. Skin tensile properties revisited during ageing. Where now, where next? J Cosmet Dermatol. 2004;3:35–40. 22. Henry F, et al. Age-related change facial skin contours and rheology. J Am Geriatr Soc. 1997;45:220–222. 23. Arora S, et al. Estrogen improves endothelial function. J Vasc Surg. 1998;27:1141–1146. 24. Lim SC, et al. The effect of hormonal replacement therapy on the vascular reactivity and endothelial function of healthy individuals and individuals with type 2 diabetes. J Clin Endocrinol Metab. 1999;84:4159–4164. 25. Quatresooz P, et al. Immunohistochemical clues at aging of the skin microvascular unit. J Cutan Pathol. 2008, Epub June 17 (2008). 26. Margolis DJ, Knauss J, Bilker W. Hormone replacement therapy and prevention of pressure ulcers and venous leg ulcers. Lancet. 2002;359:675–677.
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27. Pie´rard-Franchimont C, et al. Skin water-holding capacity and transdermal estrogen therapy for menopause: a pilot study. Maturitas. 1995;22:151–154. 28. Paquet F, et al. Sensitive skin at menopause; dew point and electrometric properties of the stratum corneum. Maturitas. 1998;28: 221–227. 29. Le´veˆque JL, Gubanova E. Influence of age on the lips and perioral skin. Dermatology. 2004;208:307–313. 30. Caisey L, et al. Influence of age and hormone replacement therapy on the functional properties of the lips. Skin Res Technol. 2008;14: 220–225. 31. Roux C. Randomised, double-masked, 2 years comparison of tibolone with 17b estradiol and norethindrone acetate in preventing postmenopausal bone loss. Osteoporosis Int. 2002;13:241–248.
32. Sator PG, et al. The influence of hormone replacement therapy on skin ageing: a pilot study. Maturitas. 2001;39:43–55. 33. Pie´rard-Franchimont C, Pie´rard GE. Post-menopausal aging of the sebaceous follicle. A comparison between women receiving hormone replacement therapy or not. Dermatology. 2002;204:17–22. 34. Castello-Branco C, et al. Relationship between skin collagen and bone changes during aging. Maturitas. 1994;18:199–206. 35. Shah MG, Maibach HI. Estrogen and skin : an overview. Am J Clin Dermatol. 2001;2:143–150. 36. Pie´rard GE, et al. Relationships between bone mass density and tensile strength of the skin in women. Eur J Clin Invest. 2001;31:731–735.
18 Cluster of Differentiation 1d (CD1d) and Skin Aging Mohamed A. Adly . Hanan Assaf . Mahmoud R. Hussein
Introduction CD1d is a member of CD1 family of transmembrane glycoproteins which represent a third lineage of antigenpresenting molecules. These molecules are distantly related to the classical major histocompatibility complex (MHC) molecules in the immune system [1–4]. However, unlike the first and second lineages of antigen-presenting molecules (the classical MHC class I and class II molecules) that bind peptide antigens, CD1 molecules have evolved to bind lipids and glycolipids [5–7]. CD1 family molecules are closely related to MHC class Ia and Ib proteins by sequence homology, domain organization (a1, a2, a3, and b2m), and association with b2 microglobulin rather than to class II molecules [2, 6]. In contrast to MHC class I molecule which is polymorphic, CD1 molecules are not polymorphic [1–3], and are encoded by linked genes outside the MHC complex; the gene for CD1d is located on chromosome 1 in humans [1–4]. The CD1 family is divided into two groups by sequence homology: group I which consists of CD1a, -b and -c isotypes and groupII which includes CD1d [8]. Only the group II CD1d isotypes are preserved in human, mouse, rat, rabbit, and monkey [4, 9]. Sequence similarity is substantially higher for the same isotype from different species than for different isotypes within the same species [1–3, 10], suggesting that each group of CD1 molecules could have a different function [4]. CD1d binds glycol-and phospholipid antigens, and is essential for the development and activation of a subset of T cells known as natural killer T (NK-T) cells which are characterized by the expression of receptors used by NK cells [1–3, 6, 11] and invariant Va-Ja TCRs, such as Va24JaQ TCR in humans and Va14Ja281 TCR in mice [12]. NK-T cells recognize self or nonself glycolipids presented by CD1d molecule, and respond by secretion of cytokines; most notably IFN-g and IL-4 [1–3, 6, 13]. The synthetic glycolipid molecule a-galactosylceramide (a-GalCer) was shown to stimulate human and mouse NK-T cells in a CD1d-restricted manner [14–16]. CD1d plays,
therefore, via the production of cytokines secreted by NK-T cells, a critical role in performing a number of immunoregulatory functions within the human and mammalian body including protection against autoimmune diseases, microbial infection, and cancer. In mice, it was shown that CD1d regulates UV-induced carcinogenesis by inhibiting apoptosis to prevent elimination of potentially malignant keratinocytes and fibroblasts [17, 18].
CD1d Expression in the Human Skin Recently, CD1d expression and NK-T cells were demonstrated in the epidermis of acute and chronic psoriatic plaques [19–22]. Not only did CD1d show expression in psoriatic skin, but also in normal sun protected and scalp skin [22–25]. Moreover, it was found that CD1d is expressed on human scalp hair follicle keratinocytes, and that its expression undergoes hair cycle-associated changes, suggesting a role in hair follicle cycle regulation.
CD1d Expression in Human Skin Undergoes Age-Associated Changes The expression pattern of CD1d in different age groups was examined recently and variable profiles found [25]. CD1d had a strong expression in the skin of young (between 6–18 year old), but it declined in the skin of mid (between 30–40 year old) and old (50–81) age groups. In the epidermis, CD1d was expressed in all layers except in the stratum corneum (> Figs. 18.1–18.5). However, its expression had different intensities, with strong immunoreactivity in the stratum basale and stratum spinosum but weak immunoreactivity in the stratum granulare, particularly in old individuals (Table 18.1). The findings of age-related decrease of CD1d protein expression in the human epidermis agree with other groups [26, 27]. Decreased density and function of
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_18, # Springer-Verlag Berlin Heidelberg 2010
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Cluster of Differentiation 1d (CD1d) and Skin Aging
. Figure 18.1 Immunoreactivity of CD1d protein in human skin of 6 (A), 15 (B), 18 (C), 33 (D), 39 (E), 53 (F), 57 (G), 60 (H), 68 (I), 71 (J), 78 (K) and 81 (L) year old individuals, shown in red color with TSA technique. M negative control; N positive control shows CD1d expression in a blood vessel. At the age of 6 years, CD1d immunoreactivity was not only strong, but also seen in all layers of the epidermis except the stratum corneum (A). In the ages between 10 and 30 year old, CD1d immunoreactivity decreased, and was detected in the Malpighian layer, stratum spinosum, and a few layers of stratum granulosum (B–C). In the ages between 30 and 40 year old, CD1d had moderate expression that was mainly seen in the stratum basale (D–E). In the ages between 40 and 60 years, CD1d protein expression was moderate immunoreactivity, and localized to the basal and granular layers (F). In the skin derived from 57 year old donors, the immunoreactivity was however stronger, and the expression was apparent both in the basal and granular layers (G). In the old ages, the expression of CD1d was confined to the stratum basale (H). Sometimes, CD1d protein expression was seen in the stratum spinosum and some cells of the stratum granulosum, in addition to the basal layer (I–L). Reprinted with permission from Adly et al. [23, 25] ß 2005, 2006, Wiley-Blackwell
. Figure 18.2 Immunoreactivity of CD1d protein in human skin of 6 (A), 18 (B), 33 (C), 53 (D), 60 (E) and 71 (F) year old individuals, shown in red color with ABC technique. At the age of 6 years, CD1d immunoreactivity was not only strong but also seen in all layers of the epidermis except the stratum corneum (A). In the ages between 10 and 30 year old, CD1d immunoreactivity decreased, and was detected in the Malpighian layer, stratum spinosum and a few layers of stratum granulosum (B). In the ages between 30 and 40 year old, CD1d had moderate expression that was mainly seen in the stratum basale (C). In the ages between 40 and 60 years, CD1d protein expression was moderate immunoreactivity and localized to the basal and granular layers (D). Reprinted with permission from Adly et al. [23, 25] ß 2005, 2006, Wiley-Blackwell
Cluster of Differentiation 1d (CD1d) and Skin Aging
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Cluster of Differentiation 1d (CD1d) and Skin Aging
. Figure 18.3 Immunoreactivity of CD1d protein in sweat glands of human skin derived from 6 (A), 33 (B), 39 (C), 64 (D), 71 (E), and 78 (F) years old individuals, shown in red color with TSA technique. CD1d protein expression was strong in sweat glands of all ages. Reprinted with permission from Adly et al. [23, 25] ß 2005, 2006, Wiley-Blackwell
Cluster of Differentiation 1d (CD1d) and Skin Aging
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. Figure 18.4 Immunoreactivity of CD1d protein in sebaceous glands of human skin derived from 15 (A), 39 (B) and 71 (C) year old individuals, shown in red color with TSA technique. In sebaceous glands, CD1d protein expression was strong in all ages. Interestingly, CD1d protein was comparable to that in the epidermis, i.e., it was strong in children, moderate in young adults, and weak with aging. Reprinted with permission from Adly et al. [23, 25] ß 2005, 2006, Wiley-Blackwell
. Figure 18.5 Immunoreactivity of CD1d protein in different ages. Reprinted with permission from Adly et al. [23, 25] ß 2005, 2006, Wiley-Blackwell
. Table 18.1 CD1d protein expression in the normal human skin: all the specimens were batch-stained in the same run Age groups (Years)
Stratum basale
Stratum spinposum
Stratum granulare
Stratum corneum
Dermis
6–18
2.6 0.3
2.0 0.5
1.7 0.3
0.0
2.6 0.3
30–40
2.4 0.2
1.6 0.3
1.2 0.2
0.0
2.6 0.3
50–81
1.5 0.3
1.25 0.2
0.75 0.2
0.0
2.7 0.3
The immunostaining experiments were repeated at least three times. The staining results were examined by the authors and were scored as (–) for absent, (1) for weak, (2) for moderate, and (3) for intense staining, following other groups
epidermal dendritic cell populations were found in aged C57BL/6J mice. However, the capacity of the dendritic cells to transport antigen from the skin to the draining lymph nodes was found in vivo to be comparable to that
of young mice. The strong expression of CD1d protein in the skin of children and young individuals may be due to increased recruitment, tissue accessibility, and local proliferatioin of CD1d + cells (> Table 18.1). Molecular
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Cluster of Differentiation 1d (CD1d) and Skin Aging
signaling of three distinct pathways of apoptosis, namely the death receptor pathway, the mitochondrial pathway, and the endoplasmic reticulum stress pathway may be involved in CD1d + cells apoptosis [28–31].
Conclusion CD1d plays, therefore, via the production of cytokines secreted by NK-T cells, a critical role in performing a number of immunoregulatory functions within the human and mammalian body including protection against autoimmune diseases, microbial infection, and cancer. In mice, it was shown that CD1d regulates UV-induced carcinogenesis by inhibiting apoptosis to prevent elimination of potentially malignant keratinocytes and fibroblasts.
References 1. Porcelli SA. The CD1 family: a third lineage of antigen-presenting molecules. Adv Immunol. 1995;59:1–98. 2. Porcelli SA, Segelke BW, Sugita M, Wilson IA, Brenner MB. The CD1 family of lipid antigen-presenting molecules. Immunol Today. 1998;19(8):362–368. 3. Porcelli SA, Modlin RL. The CD1 system: antigen-presenting molecules for T cell recognition of lipids and glycolipids. Annu Rev Immunol. 1999;17:297–329. 4. Zeng Z, Castano AR, Segelke BW, Stura EA, Peterson PA, Wilson IA. Crystal structure of mouse CD1: An MHC-like fold with a large hydrophobic binding groove. Science. 1997;277 (5324):339–345. 5. Hong S, Scherer DC, Singh N, Mendiratta SK, Serizawa I, Koezuka Y, Van Kaer L. Lipid antigen presentation in the immune system: lessons learned from CD1d knockout mice. Immunol Rev. 1999;169:31–44. 6. Exley M, Garcia J, Balk SP, Porcelli S. Requirements for CD1d recognition by human invariant Valpha24 + CD4-CD8- T cells. J Exp Med. 1997;186(1):109–120. 7. Sidobre S, Kronenberg M. CD1 tetramers: a powerful tool for the analysis of glycolipid-reactive T cells. J Immunol Methods. 2002;268 (1):107–121. 8. Calabi F, Jarvis JM, Martin L, Milstein C. Two classes of CD1 genes. Eur J Immunol. 1989;19(2):285–292. 9. Kashiwase K, Kikuchi A, Ando Y, Nicol A, Porcelli SA, Tokunaga K, Omine M, Satake M, Juji T, Nieda M. Koezuka Y. The CD1d natural killer T-cell antigen presentation pathway is highly conserved between humans and rhesus macaques. Immunogenetics. 2003;54 (11):776–781. 10. McMichael AJ. Lymphocytes. 1. Function. Genetic restrictions in the immune response. J Clin Pathol Suppl (R Coll Pathol). 1979; 13:30–38. 11. Brossay L, Chioda M, Burdin N, Koezuka Y, Casorati G, Dellabona P, Kronenberg M. CD1d-mediated recognition of an
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alpha-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution. J Exp Med. 1998;188 (8):1521–1528. Dellabona P, Padovan E, Casorati G, Brockhaus M, Lanzavecchia A. An invariant V alpha 24-J alpha Q/V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4–8- T cells. J Exp Med. 1994;180(3):1171–1176. Fujii S, Shimizu K, Steinman RM, Dhodapkar MV. Detection and activation of human Valpha24 + natural killer T cells using alphagalactosyl ceramide-pulsed dendritic cells. J Immunol Methods. 2003;272(1–2):147–159. Spada FM, Koezuka Y, Porcelli SA. CD1d-restricted recognition of synthetic glycolipid antigens by human natural killer T cells. J Exp Med. 1998;188(8):1529–1534. Kawano T, Cui J, Koezuka Y, Toura I, Kaneko Y, Motoki K, Ueno H, Nakagawa R, Sato H, Kondo E, Koseki H, Taniguchi M. CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides. Science. 1997;278(5343):1626–1629. Nieda M, Nicol A, Koezuka Y, Kikuchi A, Takahashi T, Nakamura H, Furukawa H, Yabe T, Ishikawa Y, Tadokoro K, Juji T. Activation of human Valpha24NKT cells by alpha-glycosylceramide in a CD1drestricted and Valpha24TCR-mediated manner. Hum Immunol. 1999;60(1):10–19. Matsumura Y, Moodycliffe AM, Nghiem DX, Ullrich SE, Ananthaswamy HN. Resistance of CD1d / mice to ultraviolet-induced skin cancer is associated with increased apoptosis. Am J Pathol. 2004; 165(3):879–887. Matsumura Y, Moodycliffe AM, Nghiem DX, Ullrich SE, Ananthaswamy HN. Inverse relationship between increased apoptosis and decreased skin cancer in UV-irradiated CD1d / mice. Photochem Photobiol. 2005;81(1):46–51. Nickoloff BJ, Wrone-Smith T, Bonish B, Porcelli SA. Response of murine and normal human skin to injection of allogeneic blood-derived psoriatic immunocytes: detection of T cells expressing receptors typically present on natural killer cells, including CD94, CD158, and CD161. Arch Dermatol. 1999;135 (5):546–552. Nickoloff BJ, Wrone-Smith T. Injection of pre-psoriatic skin with CD4 + T cells induces psoriasis. Am J Pathol. 1999; 155(1):145–158. Nickoloff BJ, Bonish B, Huang BB, Porcelli SA. Characterization of a T cell line bearing natural killer receptors and capable of creating psoriasis in a SCID mouse model system. J Dermatol Sci. 2000;24 (3):212–225. Bonish B, Jullien D, Dutronc Y, Huang BB, Modlin R, Spada FM, Porcelli SA, Nickoloff BJ. Overexpression of CD1d by keratinocytes in psoriasis and CD1d-dependent IFN-gamma production by NK-T cells. J Immunol. 2000;165(7):4076–4085. Adly MA, Assaf HA, Hussein M. Expression of CD1d in human scalp skin and hair follicles: hair cycle related alterations. J Clin Pathol. 2005;58(12):1278–1282. Adly MA, Assaf HA, Nada EA, Soliman M, Hussein M. Human scalp skin and hair follicles express neurotrophin-3 and its high-affinity receptor tyrosine kinase C, and show hair cycle-dependent alterations in expression. Br J Dermatol. 2005;153(3):514–520. Adly MA, Assaf HA, Hussein MR, Neuber K. Age-associated decrease of CD1d protein production in normal human skin. Br J Dermatol. 2006;155(1):186–191. Sunderkotter C, Kalden H, Luger TA. Aging and the skin immune system. Arch Dermatol. 1997;133(10):1256–1262.
Cluster of Differentiation 1d (CD1d) and Skin Aging 27. Gilchrest BA, Murphy GF, Soter NA. Effect of chronologic aging and ultraviolet irradiation on Langerhans cells in human epidermis. J Invest Dermatol. 1982;79(2):85–88. 28. Fainboim L, Salamone Mdel C. CD1: a family of glycolypid-presenting molecules or also immunoregulatory proteins? J Biol Regul Homeost Agents. 2002;16(2):125–135. 29. Fenske NA, Lober CW. Structural and functional changes of normal aging skin. J Am Acad Dermatol. 1986;15(4 Pt 1):571–585.
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30. Fenske NA, Conard CB. Aging skin. Am Fam Physician. 1988;37 (2):219–230. 31. Sprecher E, Becker Y, Kraal G, Hall E, Harrison D, Shultz LD. Effect of aging on epidermal dendritic cell populations in C57BL/6J mice. J Invest Dermatol. 1990;94(2):247–253.
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16 Considerations for Thermal Injury: The Elderly as a Sensitive Population Donald L. Bjerke
Introduction As the ‘‘baby boom population’’ in North America ages, one of the populations at greatest risk of thermal injury continues to expand. According to the US Census Bureau in 2000, the population of US citizens who are 75 years and older was 16,548,000 (6.0%). By 2010 this figure is projected to be 19,101,000 (6.4%), and by 2050 it is expected to exceed 54, 094,000 (13.4%) [1]. Annually, in the United States and Canada, 1.25 million people suffer burn injuries [2]. Populations identified at increased risk of burns include infants and young children, older adults, and people with any type of disability [3–6]. Many of the burns reported are from scalds. Scald injuries are painful, require prolonged treatment, and may result in lifelong scarring and even death. Most burn injuries happen in the home with tap water scalds occurring in the bathroom or kitchen [3, 7, 8]. Burns can also be caused by therapies in medical treatment facilities [9] or from therapeutic use of heat in the home. This premise is supported by a jointly issued public health advisory in 1995 by the United States Food and Drug Administration and the Consumer Products Safety Commission on electric heating pads. This advisory reported approximately 1,600 heating pad burns treated in the emergency room annually and that approximately 45% of those patients were over 65 years of age [10]. The American Burn Association classifies the severity of a burn based on the total body surface area (TBSA) affected and the depth of the injury (> Table 16.1). The elderly are at greater risk of thermal injury, and the outcome of that injury can be devastating. Elderly subjects have a higher mortality than younger subjects who have a similar surface area burn. For example, if half the body surface is burned in a young adult, the mortality is about 50%, whereas a burn of only one fifth of the body surface in the elderly results in a similar mortality [1, 8]. This increased risk in the elderly is due to many factors as a result of both physical and physiologic differences seen in this population. Diminished senses,
impaired mental acuity, slower reaction time, reduced mobility, and bedridden states may lead to the decreased ability of the elderly to identify the severity of the situation, as well as their capacity to escape from harm. Physiologic factors include thinner skin, reduced microcirculation, increased susceptibility to infections in the elderly, and higher incidence of premorbid conditions such as chronic disease, alcoholism, medications, senility, and neurological or psychiatric disorders [3]. This, in turn, may lead to an increased total body surface area burn, deeper burns, and more devastating consequences from thermal injury. This chapter discusses the conditions by which thermal injury occurs and the physiologic factors associated with an increased risk in the elderly population.
. Table 16.1 Classification of Burn Injuries Major: burn injuries Second-degree burns over a body surface area (BSA) greater than 25% in adults or 20% in children; all third-degree burns over a BSA of 10% or greater; all burns involving hands, face, eyes, ears, feet, and perineum; all inhalation injuries; electrical burns; complicated burn injuries involving fractures or other major trauma; and burns on all high-risk patients (i.e., those who are elderly or who have debilitating diseases) Moderate: uncomplicated burn injuries Second-degree burns over a BSA of 15–25% in adults or 10–20% in children; third-degree burns over a BSA of 2–10%; and burns not involving eyes, ears, face hands, feet, or perineum Minor: burn injuries Second-degree burns over a BSA of 15% or less in adults or 10% or less in children; third-degree burns over a BSA of less than 2%; and burns not involving eyes, ears, face, hands, feet, or perineum. Minor burns exclude electrical injuries, inhalation injuries, and burns on all high-risk patients
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_16, # Springer-Verlag Berlin Heidelberg 2010
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Background Information on Thermal Injury The first significant research in the area of thermal injury was conducted by Henriques and Moritz at the Harvard Medical School [11–15] in the 1940s. Prior to this work, remarkably little information existed concerning the mechanism by which hyperthermia leads to irreversible cellular injury, the reciprocal relationships of time and temperature in the production of either cutaneous or systemic injury, the relationship between environmental heat, surface temperature, and the slope of the transcutaneous thermal gradient, the pathogenesis of cutaneous burns, or the physiological mechanisms by which external heat may be responsible for acute disability and death. This research provided information on parameters controlling the flow of heat into the skin and the importance of heat capacity and thermal conductivity and developed an approximate first-order Fourier’s law equation to describe the transient heat flow. In vivo factors that affect skin temperature include site variations in the respective thickness of epidermis, dermis, fat, and muscle; variation of existing temperature gradients within the skin with respect to time and/or position of site; average rate of blood flow through the various skin layers, and variations of the rate of flow with respect to position of site and temperatures within the site; and the appearance of edema fluid in variable quantities. These factors result in site-specific alterations in the density, heat capacity, thickness, and thermal conductivity of the various layers of skin so affected. Using a pig model (skin similar to humans), Henriques and Moritz brought and held constant the skin at various temperatures until the threshold of irreversible injury occurred. From this, they derived the time–temperature relationship in the layer of basal epidermal cells, which are thought to be the most important cell layer for the production of epidermal injury by heat. Cell death (necrosis) is a result of irreversible thermal denaturation of the protein present within the cell [14, 16]. Because second- and third-degree burns involve cell death at the basal epidermal layer, the distance from the surface of the skin to this basal epidermal layer becomes important for the rate of heat transfer. In other words, a thinner epidermis results in more efficient heat transfer from the surface of the skin to the basal epidermal layer, thus increasing the risk of thermal injury. The in vitro work in pigs was extended to in vivo thermal injury in both pigs and humans. Circulating water at various temperatures was brought in contact with skin on the ventral forearms or anterior thoracic region of presumed young healthy military men. Of particular note is that the time–temperature relationship is not linear and the rate at which burning
occurs is almost doubled for each degree rise in temperature between 44 C and 51 C. Discomfort in the form of a stinging sensation occurred between 47.5 C and 48.5 C and was variable between subjects with respect to intensity. For example, severe burns were sustained without discomfort at 47 C while in other cases intense discomfort was noted before irreversible injury at temperatures above 48 C. The lowest temperature resulting in cutaneous burning was 44 C and the time required to cause irreversible damage to epidermal cells at this temperature was approximately 6 h. Alternatively, a surface temperature of 70 C resulted in trans-epidermal necrosis in less than 1 s. The relationship between temperature and duration of exposure to the extent of skin damage was landmark and has served as a guide for all subsequent works. Wu extended the work of Moritz and Henriques by adding the heat transfer reaction for a source of high energy [16]. His treatment, assuming contact between two semi-infinite bodies of finite thermal inertia at different temperatures, showed that sources of low inertia (e.g., wood, insulation, some plastics) cause a slower rise in skin temperature than a source of high thermal inertia (e.g., steel and aluminum) at the same temperature. This is explained by high thermal inertia materials, which can make more energy available at the surface in a given time than those of lesser thermal inertia. ASTM published a document entitled Standard Guide for Heated System Surface Conditions that Produce Contact Burn Injuries in October 2003 [17]. Included in this guide is a summary of the 1947 research of Moritz and Henriques. The review notes that the earlier work neglected the flow of blood to carry heat away and the physiological changes in skin properties as the damaged zone traverses the outer skin layers. Factors that increase the complexity of predicting burns include: (a) site variations with respect to the thickness of different skin layers; (b) variations of initial conditions within the skin with respect to time, position, and physical condition of the subject; (c) the unknown average rate of blood flow through the skin layers and variations within the layers with respect to location and ambient temperatures (warm ambient causes increased flow near surface and cold ambient results in less flow near surface); and (d) the appearance of watery fluids in variable quantities upon exposure that result in alterations of skin density, heat capacity, thickness, and thermal conductivity. The guide is meant to serve as an estimation of the exposure to which an average individual might be subjected and does not assume to be inclusive of unusual conditions of exposure, physical health variations, or nonstandard ambient condition. The guide applies to contact with heated surfaces only. Importantly, > Fig. 16.1 demonstrates the
Considerations for Thermal Injury: The Elderly as a Sensitive Population
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. Figure 16.1 Temperature–time relationship for burns (ß ASTM International [17]. Reprinted with permission.)
relationship between skin temperature and time as it relates to thermal injury. The following equations were developed and are reported in the document: TA ¼ 15:005 þ 0:51907 Lnðtime 1000Þ þ 352:97=½Lnðtime 1000Þ TB ¼ 39:468 0:41352 Lnðtime 1000Þ þ 190:60=½Lnðtime 1000Þ where: TA = critical contact temperature for complete transepidermal necrosis ( C) TB = critical contact temperature for reversible epidermal injury ( C) time = elapsed contact time (s) Ln = natural logarithm Exposures below the lower curve should not produce permanent injury in normal humans. Exposure between the curves are described as second-degree burns and have intermediate levels of cell damage. Exposures at levels above the top line are defined as third-degree burns that cause deep, permanent cell damage and scarring.
Reported Burn Injuries in the Elderly Barillo investigated burn injuries in medical treatment facilities [9]. The medical records of 4,510 consecutive admissions to one burn center were reviewed and a cohort
of 54 patients had suffered burn injuries as a result of medical therapy. A number of burns in the home resulted from therapeutic applications of heat, including six patients burned by heating pads, one patient burned by a heat lamp, and four patients burned by contact with hot water bottles or soaks. The average hospital stay for burns resulting from medical therapy (22.9 days) was excessive in comparison with the average total burn size of 3.0% TBSA. In addition to medical therapies, there were two patients, including one fatality, that were scalded while being bathed in nursing homes. The total body surface area (TBSA) burned by the scalding water was 20.3% with third-degree burns on 3% of the body. Contributing factors to thermal injury were advanced age, chronic illness, limited mobility, and altered skin sensation. More recently, Ghods and colleagues published the results of a survey of 36 burn clinics in Germany with regard to hot air sauna burns [18]. In total, 14 patients were treated in the German burn units between 1999 and 2005 and an additional two patients in the author’s clinic. Of note, the average age of the individuals was 67 years and the time spent in the sauna was between 45 and 60 min. In all cases, unconsciousness occurred and was assumed to be a result of orthostatic dysregulation. Deep second- and third-degree burns of the highest exposed body parts were found in all cases (an average of 14% TBSA). In two cases, the involved lower extremities had to be amputated; in four cases, primary amputations of toes or fingers were necessary, and four patients died because
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of multiple system organ failure. Normally, exposure to temperatures up to 120 C in dry, hot air do not cause damage to the skin, because of the low thermal capacity of the air. In the cases presented by the Ghods report, a hypothesis was developed to support a theory for ‘‘apex burns.’’ Unconsciousness leads to a declension of the perfusion of the skin with degraded cardiac achievement and low blood pressure. This effect, in turn results in insufficient cooling of the skin. These two reports clearly demonstrate that conditions that normally do not cause harm in a young healthy population can result in significant thermal injury to older individuals, especially when there are other concomitant factors involved. Because of the medical significance of burns in the elderly, one encouraging trend is the shift from burn treatment to burn prevention. Behavioral changes include testing water temperature before taking a bath or shower, avoidance of wearing loose sleeves while cooking, having carbon monoxide detectors that are tested regularly, and having smoke detectors that are tested regularly [3]. Additional work by Redlick and colleagues has targeted different mass media channels to promote campaigns targeted at the prevention of burns in the elderly. This groundbreaking work by the Burn Centre in Toronto along with Sunnybrook and Women’s College Health Science Centre and the Toronto Western Hospital has led to an effective burn prevention campaign for older adults [19]. Incorporated into the prevention campaign were previous success stories for lowering hot water heater settings [20] and preventing cigarette burns and contact burns with household radiator heaters [21]. While the campaign was effective at improving burn prevention knowledge, whether this results in a change in burn prevention behavior remains unclear.
Risk Factors for Thermal Injury It is well accepted that elderly individuals are at greater risk of thermal injury. Physiological factors that contribute to an increased risk include the thinning of the skin, a compromised ability to dilate the vasculature of the skin, and a reduced thermal sensitivity with advancing age. The significance of these changes are that older adults have thinner skin than their younger counterparts, so contact with a hot surface or liquid can cause deeper burns with even brief exposure. To maintain a safe skin temperature upon thermal challenge, the body mobilizes the blood circulation to the periphery to ‘‘wick away’’ the heat by acting as a convective heat exchanger [22]. With aging comes a reduction in the ability to mobilize the circulation to the periphery. In addition, the compromised ability to feel heat may be decreased with aging due
to certain medical conditions or medications so that the elderly may not realize the thermal insult (e.g., scalding bath or kitchen water) is too hot until injury has occurred. Physical conditions may also contribute to the increased risk of thermal injury. Some older adults have conditions that make them more prone to falls in the bathtub or shower or while carrying hot liquids [7]. The physical factors related to thermal injury are outside the scope of this chapter. The sections that follows will describe in more detail the literature regarding thinning of skin, compromised microcirculation, and reduced thermal sensitivity that accompany aging.
Thinning Skin For additional information on the effects of aging on skin structure, please see the other relevant sections of this book. The skin undergoes several structural and functional changes with advancing age. With regard to thermal injury, the significance of thinning of the skin is that heat applied to the surface can more easily be conducted to the basal layer of the epidermis and beyond because it has less distance to penetrate. Thus, the depth of the thermal injury can be greater in elderly than in younger individuals exposed to the same temperature. While the thinning of the skin with age has been characterized, there remains an opportunity to examine the effects of changes in skin composition that occur with increasing age in relation to thermal capacity, density, and thermal conductance. In addition, elderly skin is also more prone to blistering from mechanical sheer force, and while controversial, this effect may be exacerbated by thermal challenges. The process of aging skin is often divided into two components: intrinsic aging, which is genetically determined and extrinsic aging, which is associated with cumulative damage by UV exposure. Extrinsic aging associated with excess exposure to ultraviolet light is characterized by loss of elasticity, increased roughness and dryness, irregular pigmentation, and deep wrinkling. The epidermis may thin in response to atrophy and may be accompanied with changes in the proportion and/or functionality of the dermal extracellular components [23]. Although there are differences in intrinsic and extrinsic skin aging, it is becoming evident that there are many consistent changes at the molecular level. Changes seen with intrinsic aging such as decreased cellular lifespan, reduced response to growth factors, disruption of matrix synthesis, and elevation of proteolytic activity are all evident in photo-damaged skin. The changes are simply more pronounced [23]. Montagna and Carlisle describe aging skin with the undersurface of the epidermis becoming flattened, with
Considerations for Thermal Injury: The Elderly as a Sensitive Population
little apparent change to the epidermis except for minor alterations in the organization of its cells [24]. The dermis undergoes greater changes with aging as it diminishes in bulk, many of its collagenous elastic fiber are gradually dissolved by enzymes, and the layer of fat becomes thinner. There appears to be a steady decline in the number of fibroblasts and mast cells with advancing age. The upper dermis contains collagenous fiber bundles that are somewhat haphazardly arranged. Cerimele describes the accompanying physiological changes that occur in aging skin [25]. These include impairment of barrier function, decreased turnover of epidermal cells, reduced keratinocytes, and fibroblasts, a reduced vascular network particularly around hair bulbs and glands. These changes result in fibrosis and atrophy, and decreases in hair and nail growth, vitamin D synthesis, and the density of Langerhan cells. Reductions in the immune response, and decreased functioning of Meissner’s and Pacinian corpuscles are noted. Histological alterations of the microvasculature, including thickening of the basement membrane in the exposed areas of alterations in the veil cells in protected zones, combined with the general reduction in vasculature, are probably responsible for the gradual atrophy of the cutaneous appendages that occurs with time. All of these normal changes with aging impact on the risk of thermal injury [25]. There is also a reduction in the number and biosynthetic capacity of fibroblasts and progressive disappearance of elastic tissue in the papillary dermis. Skin collagen content decreases with age and the fine collagen fibers associated with infancy become increasingly dense and tightly packed and far more randomly oriented [23]. Martin conducted a comprehensive cadaver study of body composition with 13 un-embalmed cadavers aged 59–86 years (six male and seven female) [26]. Measurements were made at 12 sites bilaterally and one central site (abdomen). Skin thickness was measured to a precision of 0.05 mm. Skin thickness varied by subject and site with males having thicker skin than females at all individual sites and overall (1.19 mm compared to 0.96 mm). The mean of all 25 sites ranged from 0.81 to 1.43 mm in males and from 0.73 to 1.10 mm in females. The thinnest site was the bicep (0.76 mm in men and 0.49 mm in women). The thickest skin site was at the subscapular site (2.07 mm in men and 1.76 mm in women). The other body sites measured were triceps, forearm, chest, waist, supraspinal, abdominal, front thigh, medial thigh, rear thigh, patellar, and medial calf. Of note, this study determined the entire skin thickness and concluded that skin likely thins with age. Moragas and colleagues studied abdominal skin samples from 96 autopsy cases ranging in age from 3.5 months
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to 86 years [27]. Abdominal skin was chosen as shielded from photoaging and thus, the changes are attributed to intrinsic aging only. Samples, 35 mm by 15 mm, were fixed in 10% buffered formalin, subsampled and embedded in paraffin wax. Sections of 4 mm thickness were stained with hematoxylin and eosin. Four characteristics were evaluated in each case. Three were denoted by linear roughness indices: progressive flattening of the epidermal undersurface related to the rete peg profile, effect of shrinkage on the basal layer, and waviness of the interface between the granular and corneum layers due to shrinkage. The fourth variable corresponded to epidermal thickness in micrometers, measured in zones between the rete pegs. When looking at the extreme age groups (0–20 years and 80–100 years), elderly subjects had a 36% reduction in the roughness index as related to the rete peg profile as compared with younger subjects. In the elderly, the epidermis was 49.5% thinner than in younger individuals. The decrease in shrinkage indices (basal layer and waviness of the interface between the granular and corneum layers) were 6% and 22%, respectively. The average epidermal thickness for age groups 0–20, 41–60, and 81– 90 were 22.6, 17.9, and 11.4 mm, respectively. The reduction in epidermal thickness was not influenced significantly by gender [27]. The progressive decrease in mechanical resistance explains why elderly people complain that they are prone to shear-type skin injuries or show increased blistering [25]. The epidermis makes a major contribution to these changes. The two most striking epidermal features associated with aging are dermo–epidermal junction flattening, with effacement of the so-called rete ridges, and epidermal thinning [23, 27]. Epidermal tissue repair also declines with age, whether measured in terms of wound closure time or blister roof regeneration. The flattening of the dermo–epidermal junction in elderly may result in greater separation of the layers and blistering in response to shear force as compared to younger individuals. The effect of heat on pressure ulcers was investigated by Kokate and colleagues [28] in a swine model (considered to have skin similar to humans). Higher skin temperature causes an increase in tissue metabolism and oxygen consumption (about 10% for a 1 C rise). The heightened need for nutrients and oxygen cannot be fulfilled because of tissue compression resulting in ischemia and tissue damage. Kokate applied 100 mmHg of pressure and temperatures of 25 C, 35 C, 40 C, and 45 C for 5 h in the swine model. This model produced both pressure ulcers and injuries consistent with burns. At 35 C there was deep tissue damage (i.e., pressure damage), while 40 C resulted in both dermal and deep tissue damage, and 45 C caused full-thickness cutaneous and deep tissue injuries. In contrast, 25 C
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resulted in the absence of damage and was considered protective of pressure ulcers. Thus, the interplay of pressure and temperature should be considered in elderly individuals who develop injuries consistent with pressure ulcers and/or thermal injury, especially in the areas of bony prominences.
Decreased Ability for Peripheral Vasodilatation With advancing age, structural and functional changes take place in the peripheral blood vessels that result in a reduced ability to mobilize blood to the cutaneous vessels when challenged with a heat stimulus. Without the optimal convective heat exchange capacity, the skin temperature can rise more rapidly and thus shorten the time to a skin burn relative to younger individuals. This section will describe the structural and functional changes to the peripheral vasculature with age and the impact on maintaining skin temperature. In addition, studies using pharmacological actives affecting vascular tone to better understand the mechanism by which aging affects peripheral vasodilation in response to thermal stimuli will be presented. These studies will demonstrate that resting blood flow and vascular conductance decrease with age due to increased sympathetic nerve activity but during exertional heat stress, the diminished blood flow is independent of sympathetic vasoconstriction. With advancing age, there is decreased nitric oxide-mediated vasodilation and a decreased response to histamine. Thus, the reduced ability to increase blood flow to the peripheral dermal vasculature is multifactorial with effects on structure and function. Histological changes in cutaneous microvessels with age was investigated by Braverman [29]. The veil cells around normal, diabetic, and aged vessels were reconstructed in three dimensions by a computer graphics system from 120 to 140 serial ultrathin sections. While there did not appear to be any differences in the metabolic capability of the veil cells in the different groups, there were structural differences. The normal vessel was surrounded by a single layer of veil cells, which had a wrinkled and pleated surface. The veil cells around aged vessels appeared to have the same length as young veil cells but were underdeveloped in their lateral extensions so that they did not cover the vessel circumferentially as well as did the normal veil cells. Mortiz and Henriques [12] were the first to investigate the importance of blood flow with regard to the protection of the epidermis from reversible injury. Using a pig model, and applying a constant flow of water at 49 C
and 51 C for durations subthreshold to injury, increasing the pressure from 0 to 80 mmHg (thought to compress the most distal blood vessels) did not result in irreversible trans-epidermal injury. Thus, the thinness of the epidermis was considered more important than the protective effect of removing heat via the blood circulation. These early conclusions later came under scrutiny. The ASTM review notes that the increased pressure in the Moritz and Henriques study was not sufficient to collapse the blood vessels [17]. The impact of vasodilatation on protecting the surface skin temperature was subsequently demonstrated by Lipkin and Hardy [30] on the human forearm. These authors evaluated thermal inertia, which is a product of thermal conductivity, density, and thermal capacity. They noted that as heating of intact skin progressed, the influence of the increased blood flow became more pronounced, finally causing a decrease in skin temperature in spite of continued irradiation. This phenomenon did not occur when blood flow was occluded as skin temperature continued to increase. Therefore, the values of thermal conductivity, density, and thermal capacity for living skin are not constant and will depend upon blood flow, thickness of the stratum corneum, and possibly upon the state of hydration. To investigate the effects of age on the response of skin blood flow in the forearm to direct heat [31], three groups of 20 male subjects each – young (20–39 years); middleaged (40–59 years), and older (60–79 years) had blood flow measured by Doppler flowmeter. The forearm was in a water bath at 30 C that was elevated to 35 C and then 40 C. The older group demonstrated a significantly lower volume in response to 35 C and 40 C, and there was a significant reduction in blood flow for both middle-aged and older men at 40 C. Thus, aging decreases the response of cutaneous blood flow in the forearm to the direct effects of heat. The reduced blood flow suggests that this is mediated by a reduced flow response of individual microvessels in middle-aged and older men. The reduced blood volume data suggest that, in addition, vessel recruitment is depressed in older individuals. To understand the underlying physiological changes to the peripheral vasculature that occur with aging, several authors have used pharmacologic tools to investigate the sympathetic and parasympathetic nervous system. Dinenno [32] examined hemodynamic changes related to aging. Resting limb blood flow and vascular conductance are reduced with age in adult humans and these changes are related to elevations in sympathetic vasoconstrictor nerve activity and reduction in limb oxygen demands. Sixteen young males (28 1 years; mean SEM)
Considerations for Thermal Injury: The Elderly as a Sensitive Population
and 15 older males (63 1 years) were compared for femoral artery blood flow (Doppler ultrasound), vascular conductance, femoral artery resistance, and muscle sympathetic activity. Whole-limb blood flow represents the sum of flow to skeletal muscle, skin, subcutaneous tissue, and bone. Flow to subcutaneous tissue and bone is thought to be negligible at rest. Data on young adult humans in which relative measurements of whole-forearm blood flow were performed before and after skin flow was abolished with epinephrine iontophoresis suggest that skin blood flow represents 30–35% of the total flow under these conditions [33]. Femoral artery blood flow was 26% lower in the older men, despite similar levels of cardiac output. Femoral artery vascular conductance (femoral blood flow/mean arterial pressure) was 32% lower and femoral vascular resistance (mean arterial pressure/femoral blood flow) was 45% higher in older men. Muscle sympathetic nerve activity was 74% higher in the older men and correlated with femoral artery blood flow, vascular conductance, and vascular resistance. Thus, basal whole-leg arterial blood flow and vascular conductance are reduced with age in healthy adult men under resting conditions, these changes are associated with elevations in sympathetic vasoconstrictor nerve activity; and the lower wholelimb blood flow is related to a lower oxygen demand that is independent of tissue mass. The authors raise other possibilities for the findings, including a reduced bioavailability of nitric oxide with age or elevations in locally released (e.g., endothelin) or systemically circulating (e.g., vasopressin) levels of vasoconstrictor agents may have played a role. While the reduction in whole-limb blood flow of older adults at rest is thought primarily to involve differences in skeletal muscle blood flow, it raises the question as to how well skin blood flow can respond in terms of a thermal challenge. Evidence is accumulating that older adults are limited in their capacity to augment blood flow and vascular conductance in response to acute increases in functional demand imposed by large-muscle dynamic exercise, energy intake, and ambient heat stress. Kenney tested the hypothesis that an attenuated increase in cutaneous vascular conductance in elderly in response to local or reflex-mediated heat stress is due to an augmented or sustained noradrenergic vasoconstriction [34]. Forearm skin perfusion was measured by laser Doppler flowmetry in 15 young (22 þ 1 years) and 15 older (66 þ 1 years) men who exercised at 50% peak oxygen uptake in a 36 C environment. Blood flow was monitored in two sites, one of which was pretreated with bretylium tosylate (BT) to block the local release of norepinephrine and thus vasoconstriction. Forearm vascular
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conductance was 40–50% lower in the older adults. Decreased active vasodilator sensitivity to increasing core temperature, coupled with structural limitations to vasodilation, appears to limit the cutaneous vascular response to exertional heat stress in older subjects. At rest in a thermoneutral environment, human cutaneous vascular conductance is under the tonic influence of noradrenergic vasoconstrictor activity. During dynamic exercise in a warm environment, during which cutaneous vascular conductance can increase more than tenfold, vasoconstriction is withdrawn and the active vasodilator system is activated. For a given mean arterial pressure, under any given set of exercise and environmental conditions, the balance between vasoconstriction and vasodilation activity determines cutaneous vascular conductance. The conclusions of the study were that the diminished cutaneous vascular response in the skin of older subjects during exertional heat stress occurred independent of noradrenergic vasoconstriction. The mechanistic alterations that could explain this diminished vascular response were: (a) a relatively greater vasoconstrictor activity, (b) decreased vasodilator activity, or (c) end-organ response differences, which could be independent of efferent neural activity. The study of Kenney eliminates the first possibility and suggests that a combination of the latter two may be involved [34]. Minson extended the work on the effects of aging on the cutaneous microvasculature by investigating the role of nitric oxide (NO) and the axon reflexes in the skin blood flow response to local heating with advanced age [35]. Two microdialysis fibers were placed in the forearm skin of ten young (22 2 years) and ten older (77 5 years) men and women. Skin blood flow was measured by laser Doppler flowmeter. Both sites were heated to 42 C for 60 min while 10 mM NG-nitroL-arginine methyl ester (L-NAME) was infused throughout the protocol to inhibit NO synthase (NOS) in one site and 10 mM L-NAME was infused after 40 min of local heating in the second site. Local heating before L-NAME infusion resulted in a significantly reduced initial peak and plateau maximum vasodilation in elderly subjects. This finding suggests that healthy aging impacts the nerves that mediate the axon reflex or vascular responsiveness to the neurotransmitters released from these nerves. When NOS was inhibited after 40 min of heating, vasodilation declined to the same value in the young and older subjects. The initial peak response was significantly lower in the older subjects in both microdialysis sites. These data suggest that age-related changes in both axon reflex-mediated and NO-mediated vasodilation contribute to attenuated cutaneous vasodilator responses
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in the elderly. A diminished ability to rapidly increase skin blood flow in response to directly applied heat may make the elderly more susceptible to local tissue damage. Research by Tur has demonstrated age-related differences in skin blood flow in response to histamine administration [36]. In this study, the cutaneous microvascular response of older individuals (64–74 years) as compared to younger individuals (25–35 years) was slower to peak blood flow and took longer to decay. There were also regional differences such that peak blood flow was greater in the back of the young as compared to the forearm, while both sites were similar in the older cohort. While heat challenge should produce a vasodilatory response, Khan investigated the effect of aging on the vasculature during a cooling challenge and concluded that elderly subjects have diminished sympathetic vasoconstrictor responses [37]. This may be a significant factor contributing to thermoregulatory impairment in the elderly, thereby rendering them more susceptible to the harmful effects of cold weather. Using laser Doppler flowmetry sympathetic vascular responses in fingertip skin was evaluated. Indirect body heating was employed to minimize variability. The change in fingertip blood flow produced by inspiratory gasp and contralateral arm cold challenge was determined. The normal response is a rapid vasoconstriction with a subsequent decrease in fingertip blood flow, which returns to its pre-stimulus value. The study evaluated 28 elderly (mean age 68 years with SD of 4 years) and 20 younger subjects (mean age 26 years with SD of 5 years). Experiments were conducted in a room set at 25 C (55% relative humidity) and the subjects’ right arm was placed up to the elbow in a water bath maintained at 43 C. The increase in blood flow is directed mainly through arteriovenous anastomoses, but capillary blood flow also increases owing to raised local tissue temperature as a consequence of high shunt flow. The inspiratory gasp consisted of a sudden deep breath with the right arm in 43 C water throughout followed by a transfer of the right arm into a cold water bath at 15 C. A second experiment took place where subject bodies were placed in a temperature-controlled chamber in a room set at 25 C. The chamber was heated to 40 C to induce central dilation. The chamber was subsequently cooled to 12 C, which took approximately 10 min and was maintained for another 20 min. The time of blood flow to fall to 75%, 50%, and 25% from steady state was determined. Vasoconstrictor responses were significantly reduced in the elderly group in response to inspiratory gasp and cold challenge, although individual responses varied from normal to absent. The authors concluded that there is a 65% probability that an otherwise normal
elderly person will have a vasoconstrictor response considered abnormal for healthy young subjects. Whole-body cooling yielded similar results in that some elderly subjects demonstrated rapid vasoconstriction, whereas others responded with poor vasoconstrictor ability. Time to minimum blood flow after vasoconstriction was longer in the elderly, but only the time to 25% blood flow was statistically significant. While specific details on the location and nature of the effect is not clear, diminished vasoconstrictor responses most likely also result form general changes in sympathetic nervous function with age, since fingertip vasoconstriction produced by inspirator gasp and cold challenge is dependent upon sympathetic nervous activity. There was wide variability among elderly subjects in their response. The authors did not comment on the changes in blood flow with regard to heat challenge. The significance of the findings are that since thermal equilibrium is protected by reflex adjustments of cutaneous blood flow in the extremities, diminished vasoconstrictor response would promote significant heat loss in the elderly during cold exposure. The reduced vasoconstrictor response most probably occurs in the thermoregulatory shunts because a major proportion of the laser Doppler finger blood flow signal arise from flow through arteriovenous anastomoses.
Effects of Aging on Thermoregulation While increased morbidity and mortality in the elderly population during heat waves has been well documented in several medical reports, relatively few scientific studies have focused on the physiological basis of the aging process in thermoregulation, and those that have produced conflicting results. This section examines research on the effects of age on thermal regulation during exertion or exposure to increased ambient temperatures. Changes in the basic physiological mechanisms of thermoregulation may contribute to a decreased ability to avoid hyperthermia in the elderly. These changes may involve the ability to sweat and the vasomotor response to heat and could result from structural changes in the skin as well as less-effective neural regulation of blood flow and sweating. Weiss in 1992 examined the capillary blood flow velocity in the feet of ten young (ages 28–43) and 12 elderly (ages 72–84) men at skin temperatures of 32 C and 44 C [38]. The mean peak capillary blood flow in the elderly (102 mV measured by laser Doppler flowmetry) was lower than in the young population (163 mV). Considering that the magnitude, but not the pattern of skin perfusion varied between the groups, the authors concluded that aging is associated with the loss of capillary
Considerations for Thermal Injury: The Elderly as a Sensitive Population
plexus functional units, and therefore skin perfusion is lower in aged people [38]. Martin measured the maximal forearm skin vasodilatory capacity across a group of 74 subjects ranging in age from 5 to 85 years [39]. Maximal forearm skin vascular conductance was the endpoint of choice and represents the maximum forearm skin blood flow divided by the mean arterial blood pressure. The results demonstrated a progressive decrease in maximum forearm skin vascular conductance with age from young adulthood through old age. The authors note substantial histological and scanning electron micrographic evidence in the underside of the epidermis including the collapse, disorganization, and even total disappearance of vessels comprising the microcirculation. Others report a decrease in the number of superficial capillary loops in the skin as it ages. Such changes are consistent with the attenuated maximal skin blood flow response in older adults. However, a reduced blood flow to existing vessels cannot be ruled out as well. To evaluate the effect of age on thermoregulation Sagawa exposed six older (61–73 years of age) and ten younger (21–39 years of age) Japanese men to 40 C and 40% relative humidity (while sitting) for up to 130 min and examined sweat responses, esophageal, and skin temperatures, non-evaporative heat exchange, heart rate, cardiac output, blood pressure, forearm blood flow, and metabolic heat production [40]. There was no significant difference in sweat rate or in onset of sweating between the groups (> Table 16.2). Changes in skin temperature, non-evaporative heat exchange, metabolic heat production, heart rate, and cardiac output were the same during heat exposure in both groups. However, forearm blood flow before and after exposure to heat was significantly lower in the elderly group. These data suggest that the greater health risk posed to resting, yet healthy, aged
. Table 16.2 Effect of heat exposure (40 C and 40% relative humidity) on select hemodynamic parameters from Sagawa et al., 1988 study Age group
Control
Onset of sweating
At 95 min
Forearm blood flow (mL/100 mL/min) Elderly
1.3 0.2
6.0 1.7
4.6 0.7
Young
2.6 0.4*
7.2 0.9
8.7 1.4*
Forearm vascular conductance (mL/100 mL/min/Torr ¥ 100) Elderly
1.3 0.2
7.2 2.4
5.2 0.7
Young
3.0 0.5*
9.3 1.0*
12.2 1.9*
*Significant difference between age groups (p < 0.05)
16
men by hot environments is not a consequence of inadequate sweating but could be associated with retardation of the cutaneous vasodilatation reflex, which can prevent effective transfer of the body heat to its shell, thus resulting in greater heat storage. The impairment of vasomotor function in aged persons is not related to inadequate cardiac response, but is perhaps associated with insufficient vasoconstriction of the blood supply to the viscera, resulting in less blood being shunted to the skin. This suggestion is probable because, in old age, the responsiveness of the circulatory system to adrenergic nerve control is known to decrease [41]. Therefore, in elderly individuals, a decreased response to beta stimulation could explain the impaired vasodilatory responses, and a lowered response to alpha stimulation could be the underlying mechanism for diminished vasoconstriction in the viscera. The effects of age and acclimation on responses to passive heat were studied by Armstrong and Kenney [42]. Six older men (61 1 years) were compared to six young men (26 2 years) in an environmental chamber during a systematic increase in dry-bulb temperature from 28 C to 46 C followed by 30 min in a constant 46 C environment. If older and younger subjects are matched for VO2max, anthropometry (height, weight, and surface area-to-mass ration), and body composition (skinfold thickness and adiposity), no temperature differences are seen during a passive thermal stress of this magnitude. The authors note that previous studies concluded that older subjects respond to passive heat stress with greater elevations in core temperature than young subjects of the same gender, although this is not a universal finding. Much of the discrepancy can be attributed to subject selection criteria, i.e., such factors as body surface area-to-mass ratio, adiposity, and especially VO2max. The control of heat-induced cutaneous vasodilatation in relation to age was determined in subjects of 55–68 years of age as compared to subjects of 19–30 years of age [43]. Subjects performed 75 min cycle exercises in a hot environment (37 C, 60% relative humidity). Core body temperature and skin temperature rose to the same level in both groups and there were no differences in rate of sweating. However, older subjects responded with lower arm blood flow by about 40% as compared to the younger counterparts. These results suggest an altered control of skin vasodilatation during exercise in the heat in older individuals. Havenith extended this work by examining the relative influence of age (ranging from 20 to 73 years of age) on cardiovascular and thermoregulatory responses to low intensity cycle exercise (60 W for 1 h) in a warm humid environment (35 C, 80% relative humidity) [44]. The results suggest that age is an important contributory
167
168
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Considerations for Thermal Injury: The Elderly as a Sensitive Population
factor in cardiovascular effector responses to a humid heat stress test, in particular, for heart rate and skin blood flow (forearm blood flow and forearm vascular resistance), both lower with advancing age. Again, there were no age-related effects on sweating rate. In the warm humid climate chosen for the experiment, in which both dry and evaporative heat loss are minimal, the effect of a reduced skin blood flow on core temperature is likewise minimal. During exercise in warm environments, as core temperature rises, skin blood flow increases to facilitate the convective transfer of heat from core to skin. Both the slope of the skin blood flow–core temperature relationship and the steady-state skin blood flow achieved are attenuated in older subjects. It has been hypothesized that this decreased cutaneous vasodilatory response with aging is due to structural changes in the cutaneous vasculature [26]. Rooke examined maximum blood flow in elderly men and noted that blood flow does not increase as much when participants were subjected to total body heating and exercise as compared to younger adults [45]. Local heating of the forearm of seven young men (average age 31 years) and seven elderly men (average age 71 years) showed differences between the two age groups. Skin temperature was raised from 32–35 C to 42 C for 60 min. At baseline, skin blood flow in the two age groups (26–37 years and 66–82 years of age) were comparable. During the last 10 min of heating, blood flows as measured by venous plethysmography were much lower in the elderly than in the young subjects (11.1 2.7 vs 19.9 5.2 mL/min/100 mL, respectively). Thus, aging results in a reduction of the maximal conductance of the cutaneous vasculature. The authors suggest that the major limitation of skin blood flow in the elderly is intrinsic to the structure and function of the skin and not due to autonomic dysfunction. Changes in the number, size, and tortuosity of blood vessels in aged skin could be the cause of limited skin blood flow in the elderly. With age, the dermal thickness decreases by 20% and becomes relatively avascular. The implications are that the elderly appear to be less effective at maintaining normal body temperature than young adults. This applies to both cold and heat exposure, as suggested by a disproportionately high occurrence of hypothermia and heat stroke in the elderly.
Decreased Ability to Feel Heat Insensate skin and chronic illness such as diabetes mellitus are common risk factors in patients burned by therapeutic heat application [9]. The scald potential from hydrotherapy in patients with diabetic or other neuropathy is well
documented [46, 47]. A second group at risk are patients requiring cutaneous, fasciocutaneous, or myocutaneous flap procedures for surgical reconstruction. Transposed flap tissue may be insensate and may also have compromised circulation, which interferes with heat dispersal [48]. Burns have been accidentally produced in anesthetized, unconscious, or immobilized patients by the use of hydrotherapy, heating blankets, hot water bottles, or other warming devices both within the hospital and in the field by Emergency Medical Services. Similarly, the elderly have been identified as a population at greater risk of thermal injury secondary to decreased sensory perception and having a higher threshold for pain [25]. This demonstrates the importance of the ability to sense noxious thermal stimuli so as to remove one self from the burn hazard before extensive damage occurs. Pacinian and Meissner’s corpuscles, which are responsible for pressure and superficial tactile perception, respectively, undergo progressive disorganization and histological degeneration, possibly accompanied by functional loss, with advancing age. Free nerve endings do not seem to be substantially modified. While Buettner [49] reports on the effects of radiant and direct contact heat being similar with a pain threshold at 44.8 C, this work was done with a small group (N ¼ 5) presumably in the general population. Lautenbacher and Strian [50] studied the thresholds for heat pain in 64 healthy persons from 17 to 63 years of age (32 women and 32 men). The stimuli were applied to the thenar and the dorsum pedis with a contact thermode. The thresholds increased significantly with age for the foot, but not the hand. The length of the afferent pathways seems to influence the degree of agerelated changes both in heat-pain perception and in thermal sensitivity, resulting in a distal-proximal pattern of age-dependent decline (> Table 16.3). While the pain threshold for the hand did not show statistical significance with regard to age, there was a positive trend for an elevation in pain threshold with advancing age. Estimated threshold elevations based on quadratic regression curves between 15 and 65 years of age are 0.6 C on the hand and 2.2 C on the foot. The increase of threshold for determining warmth and cool on the foot (but not hand) increased significantly with advancing age. The authors note that it is unlikely that the findings of reduced heat pain and thermal sensitivity on the foot are produced only by age changes of the skin: the free nerve endings of the nociceptive and thermoceptive afferents are mainly located near the epidermal–dermal junction. The flattening of the junction and the thinning of the epidermis and dermis with increasing age may indeed result in more frequent damage to the free nerve endings, and also
16
Considerations for Thermal Injury: The Elderly as a Sensitive Population
. Table 16.3 Thresholds for detecting cool, warm, and heat pain when the stimulus is applied to the hand or foot of subjects 17 to 63 years of age, as reported by Lautenbacher and Strain, 1991 Modality
Site
Cooling sensitivity
Hand
Warming sensitivity
Pain threshold
Modality
Age 17–29 Age 30–44 Age 45–63 years years years 0.8 0.3 C
1.0 0.4 C
0.9 0.4 C
1.4 1.0 C
1.7 1.0 C
2.2 1.6 C
Handb 1.3 0.7 C
1.9 1.0 C
1.6 0.9 C
Foota
. Table 16.4 Thresholds for detecting cool, warm, and heat pain when the stimulus is applied to the chin or lip of subjects 20 to 89 years of age, as reported by Heft et al., 1996
Foota
4.5 1.9 C
6.1 2.8 C
6.2 3.2 C
Hand
45.6 2.5 C
45.2 2.5 C
45.7 1.8 C
Foota
44.9 1.5 C
44.8 1.7 C
45.7 1.2 C
Significant age effect by linear trend analysis, p < 0.05 Significant age effect by quadratic trend analysis, p < 0.05
a
b
in a decrease in the thermal resistance, which would tend to appear as heightened sensitivity. Chakour investigated the effects of age on pain perception mediated by Ad-fibers and C-fibers [51]. During preand post-nerve block periods, older adults (over 65 years of age) exhibited a significant elevation in thermal pain threshold relative to younger adults (20–40 years of age) in response to a noxious CO2 laser thermal stimulus. However, when Ad-fiber function was impaired and only C-fiber information was available, both groups responded similarly. These findings support the notion of a differential age-related change in Ad-fiber-mediated epicritic pain (phasic pain, sharp and pricking in nature) perception versus C-fiber-mediated protopathic pain (tonic pain, dull, burning, or aching in nature) with older adults have an increased thermal pain threshold (i.e., decreased pain sensitivity) as compared to younger adults. The magnitude of loss in sensitivity to mechanical stimuli is greater than to thermal stimulation and several authors have suggested that myelinated fiber function may be more prone to the effects of advancing age. Kenshalo investigated absolute thresholds for six modes of cutaneous stimulation applied to two sites in 27 young (ages 19–31) and elderly (ages 55–84) humans at the thenar eminence (hand) and the plantar foot [52]. The modes were tactile, vibration at 40 and 250 Hz, temperature increases or decreases, and noxious
Cool Warm Pain
Site Chin
Age 30 yearsa 31.5 C
Age 80 yearsa 30.8 C
Change/year ( C) 0.01
Lip
32.4 C
31.9 C
0.01
Chin
33.8 C
36.1 C
0.05
Lip
33.8 C
34.3 C
0.01
Chin
43.9 C
47.1 C
0.06
Lip
43.2 C
45.6 C
0.05
a
Predicted by linear regression
heat (via conductive heat application). For heat-pain threshold, the temperature applied was 40 C and increased at a rate of 0.3 C/s until the participant pushed a spring loaded button signifying the detection of heat pain. In this study, no statistically significant differences were found between young (mean 44.60 C and 46.46 C) and elderly (mean 44.95 C and 46.69 C) hands or feet, respectively, in their sensitivity to heat-pain stimulation. In both populations, the hands were more sensitive to a noxious heat stimulus than the feet. A larger study was conducted by Heft that evaluated 179 healthy adults aged 20–89 years who rated threshold and suprathreshold warming, cooling, and painful stimuli applied to the upper lip and chin of the face [53]. The results agree with those of Kenshalo in that while there were slight elevations in detection thresholds for cool, warm, and painful stimuli in older subjects, under suprathreshold conditions there were no statistically significant age differences for the painful stimuli (> Table 16.4). The observed threshold changes and the less consistent changes in suprathreshold performance for the non-noxious stimuli may be related to changes in peripheral nerve function, skin composition, or central nervous system function. Peripheral changes of note would include changes in the underlying innervation of the tissue, changes in the thermal conductivity of the supporting tissues, or both. Free nerve endings, which are associated with the thermal and pain sensations, remain intact into old age [54]. It can be concluded that there is a slight diminution in threshold and supra-threshold thermal performance with increasing age, and they speculate that these changes are best explained at this time by alterations in the skin thermal conductivity. Using linear regression of the data, Heft summarized the thresholds for warming, cooling, and pain conditions at the lip and chin sites in 179 subjects [53].
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Considerations for Thermal Injury: The Elderly as a Sensitive Population
Burn Prevention
Acknowledgments
One example of burn prevention is that associated with water heaters in the home. Feldman reported on unsafe bath water temperatures in the Seattle area where 80% of homes tested had bathtub water temperatures of 54 C or greater, exposing the occupants to the risk of fullthickness scalds within 30 s exposure [55]. Such burns can be prevented by limiting household water temperatures to less than 52 C. This work led to educational campaigns and legislation to lower water heater temperatures to 49–54 C [56, 57]. This work, which reduced hot water heater set points from 60–65 C down to 49 C was effective in reducing the frequency, morbidity, and mortality of tap water burn injuries. The average bath temperature of a group of 20 subjects after lowering the water heater set point was 40.5 C; with a range from 36 C to 42.5 C. Average shower temperatures (seven subjects) were slightly lower than for baths, 40 C (range 38.5– 41.0 C) [58]. These and other easy interventions in the home can reduce the risk of injury to elderly individuals.
The author expresses his appreciation and gratitude to Dr. Karen Blackburn, Dr. Rob Rapaport, and Dr. Jim McCarthy for their valuable scientific comments and suggestions.
Conclusion Thermal injuries in the general population are not uncommon. One particular population at increased risk, the elderly, continues to expand. This increased risk can be explained by several physical and physiological changes that occur with aging. These changes include thinning of the skin, reduced ability to vasodilate the peripheral vasculature in a protective response to a heat stimulus (to remove the heat from the area and maintain a safe skin temperature), and a reduced sensitivity to noxious heat stimuli with advancing age. While the landmark work of Henriques and Moritz has provided data that demonstrates the temporal relationship between skin temperature and thermal injury, additional work is needed to understand this relationship in elderly individuals. Whether advancing age shifts the time–skin–temperature curve relative to a younger population is not known. Regardless, more and more evidence demonstrate that the elderly are less able to defend against a thermal challenge relative to their younger counterparts. Because thermal injury in an elderly population can have more significant consequences with respect to morbidity and mortality, the prevention of these injuries has utmost significance. Burn prevention campaigns thus become very important and effective tools in communicating that older adults are at greater risk of thermal injury and simple changes in behavior in the home environment can prevent these injuries [3, 7, 19, 56].
References 1. Lionelli GT, Pickus EJ, Beckum OK, et al. A three decade analysis of factors affecting burn mortality in the elderly. Burns. 2005;31: 958–963. 2. Burn Foundation. Burn incidence and treatment in the United States 1999 fact sheet. Philadelphia, PA, 1999. 3. Redlick F, Cooke A, Gomez M, et al. A survey of risk factors for burns in the elderly and prevention strategies. J Burn Care Rehabil. 2002;23:351–356. 4. Baptiste MS, Feck G. Preventing tap water burns. Am J Public Health. 1980;70:727–729. 5. Petro JA, Belger D, Salzberg CA, et al. Burn accidents and the elderly: what is happening and how to prevent it. Geriatrics. 1989;44 (3):25–48. 6. Stassen NA, Lukan JK, Mizuguchi NN, et al. Thermal injury in the elderly: when is comfort the right choice? Am Surg. 2001;67: 704–708. 7. American Burn Association. Scalds: a burning issue. A campaign kit for burn awareness week, 2000. 8. Bull JP, Lawrence JC. Thermal conditions to produce skin burns. Fire Mater. 1979;3(2):100–105. 9. Barillo DJ, Coffey EC, Shirani KZ, et al. Burns caused by medical therapy. J Burn Care Rehabil. 2000;21:269–273. 10. Burlington DB, Brown A. FDA/CPSC public health advisory: hazards associated with the use of electric heating pads, 1995. http://www.fda.gov/downloads/MedicalDevices/Safety/AlertsandNotices/PublicHealthNotifications/UCM062624.pdf. 11. Henriques FC, Moritz AR. Studies of thermal injury. I: the conduction of heat to and through skin and the temperatures attained therein. A theoretical and experimental investigation. Am J Pathol. 1947;23:531–549. 12. Moritz AR, Henriques FC. Studies of thermal injury. II: the relative importance of time and surface temperature in the causation of cutaneous burns. Am J Pathol. 1947;23:695–720. 13. Moritz AR, Henriques FC, Dutra FR, et al. Studies of thermal injury. IV: an exploration of casualty-producing attributes of conflagrations; local and systemic effects of generalized cutaneous exposure to excessive circumambient (air) and circumradiant heat of varying duration and intensity. Arch Pathol. 1947;43:466–488. 14. Henriques FC. Studies in thermal injury. V: the predictability and the significance of thermally induced rate processes leading to irreversible epidermal injury. Arch Pathol. 1947;43:489–502. 15. Henriques FC. Studies of thermal injury VII. Automatic recording calorie applicator and skin tissue and skin surface thermocouples. Rev Sci Instr. 1947;18:673–680. 16. Wu Y-C. Material properties criteria for thermal safety. J Mater. 1972;1(4):573–579. 17. ASTM Designation. C 1055–03: Standard guide for heated system surface conditions that produce contact burn injuries. Published October 2003, pp. 1–8.
Considerations for Thermal Injury: The Elderly as a Sensitive Population 18. Ghods M, Corterier C, Zindel K, et al. Case report. Hot air sauna burns. Burns. 2008;34:122–124. 19. Tan J, Banez C, Cheung Y, et al. Effectiveness of a burn prevention campaign for older adults. J Burn Care Rehabil. 2004;25:445–451. 20. Katcher ML. Prevention of tap water scald burns: evaluation of a multi-media injury control program. Am J Public Health. 1987;77: 1195–1197. 21. Harper RD, Dickson WA. Reducing the burn risk to elderly persons living in residential care. Burns. 1995;21:205–208. 22. Diller KR. Analysis of burns caused by long-term exposure to a heating pad. J Burn Care Rehabil. 1991;12:214–217. 23. Jenkins G. Molecular mechanisms of skin ageing. Mech Ageing Dev. 2002;123:801–810. 24. Montagna W, Carlisle K. Structural changes in ageing skin. Br J Dermatol. 1990;122(Suppl 35):61–70. 25. Cerimele D, Celleno L, Serri F. Physiological changes in aging skin. Br J Dermatol. 1990;122(Suppl 35):13–20. 26. Martin AD. Skin thickness: caliper measurement and typical values. CRC Press, Boca Raton, 1995, pp. 293–296. 27. Moragas A, Castells C, Sans M. Mathematical morphologic analysis of aging-related epidermal changes. Anal Quant Cytol Histol. 1993;15:75–82. 28. Kokate JY, Leland KJ, Held AM, et al. Temperature-modulated pressure ulcers: a porcine model. Arch Phys Med Rehabil. 1995;76: 666–673. 29. Braverman IM, Sibley J, Keh-Yen A. A study of the veil cells around normal, diabetic, and aged cutaneous microvessels. J Invest Dermatol. 1986;86:57–62. 30. Lipkin M, Hardy JD. Measurement of some thermal properties of human tissues. J Appl Physiol. 1954;7:212–217. 31. Richardson D. Effects of age on cutaneous circulatory response to direct heat on the forearm. J Gerontol. 1989;44:M189–M194. 32. Dinenno FA, Jones PP, Seals DR, et al. Limb blood flow and vascular conductance are reduced with age in healthy humans. Relation to elevations in sympathetic nerve activity and declines in oxygen demand. Circulation. 1999;100:164–170. 33. Detry JMR, Brengelmann GL, Rowell LB, et al. Skin and muscle components of forearm blood flow in directly heated resting man. J Appl Physiol. 1972;32:506–511. 34. Kenney WL, Morgan AL, Farquahar WB, et al. Decreased active vasodilator sensitivity in aged skin. Am J Physiol. 1997;272: H1609–H1614. 35. Minson CT, Holowatz LA, Wong BJ, et al. Decreased nitric oxideand axon reflex-mediated cutaneous vasodilation with age during local heating. J Appl Physiol. 2002;93:1644–1649. 36. Tur E. Age-related regional variations of human skin blood flow response to histamine. Acta Derm Venereol (Stockh.) 1995;75: 451–454. 37. Khan F, Spence VA, Belch JJF. Cutaneous vascular responses and thermoregulation in relation to age. Clin Sci. 1992;82:521–528.
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38. Weiss M, Milman B, Rosen B, et al. Analysis of the diminished skin perfusion in elderly people by laser Doppler flowmetry. Age Ageing. 1992;21:237–241. 39. Martin HL, Loomis JL, Kenney WL. Maximal skin vascular conductance in subjects aged 8–85 yr. J Appl Physiol. 1995;79(1):297–301. 40. Sagawa S, Shiraki K, Yousef MK, et al. Sweating and cardiovascular responses of aged men to heat exposure. J Gerontol. 1988;43: M1–M8. 41. Roberts J, Steinberg GM. Effects of aging on adrenergic receptors: introduction. Fed Proc. 1986;45:40–41. 42. Armstrong CG, Kenney WL. Effects of age and acclimation on responses to passive heat exposure. J Appl Physiol. 1993;75 (5):2162–2167. 43. Kenney WL. Control of heat-induced cutaneous vasodilatation in relation to age. Eur J Appl Physiol. 1988;57:120–125. 44. Havenith G, Inoue Y, Luttikholt V, et al. Age predicts cardiovascular, but not thermoregulatory, responses to humid heat stress. Eur J Appl Physiol. 1995;70:88–96. 45. Rooke GA, Savage MV, Brengelmann GL. Maximal skin blood flow is decreased in elderly men. J Appl Physiol. 1994;77(1):11–14. 46. Katcher ML, Shapiro MM. Lower extremity burns related to sensory loss in diabetes mellitus. J Fam Pract. 1987;24(2):149–151. 47. Balakrishnan C, Rak TP, Meininger MS. Burns of the neuropathic foot following use of therapeutic footbaths. Burns. 1995;21:622–623. 48. Cavadas PC, Bonanad E. Unusual complications in a gracilis myocutaneous free flap. Plast Recontstr Surg. 1996;97:683. 49. Buettner K. Effects of extreme heat and cold on human skin. II. Surface temperature, pain and heat conductivity in experiments with radiant heat. J Appl Physiol. 1951;3:703–713. 50. Lautenbacher S, Strin F. Similarities in age differences in heat pain perception and thermal sensitivity. Funct Neurol. 1991;6:129–135. 51. Chakour MC, Gibson SJ, Bradbeer M, et al. The effect of age on Adand C-fibre thermal pain perception. Pain. 1996;64:143–152. 52. Kenshalo DR. Somesthetic sensitivity in young and elderly humans. J Gerontol. 1986;41:732–742. 53. Heft MW, Cooper BY, O’Brien KK, et al. Aging effects on the perception of noxious and non-noxious thermal stimuli applied to the face. Aging Clin Exp Res. 1996;8:35–41. 54. Montagna W, Carlisle K. Structural changes in aging human skin. J Invest Dermatol. 1979;73:15–20. 55. Feldman KW, Schaller RT, Feldman JA, et al. Tap water scald burns in children. Pediatrics. 1978;62(1):1–7. 56. Liao C-C, Rossignol AM. Landmarks in burn prevention. Burns. 2000;26:422–434. 57. Erdman TC, Feldman KW, Rivara FP, et al. Tap water burn prevention: the effect of legislation. Pediatrics. 1991;88:572–577. 58. Lawrence JC, Bull JP. Thermal conditions which cause skin burns. Inst Mech Engineers Eng Med. 1976;5:61–63.
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Stratum Corneum
36 Corneocyte Size and Cell Renewal: Effects of Aging and Sex Hormones Enzo Berardesca . Joachim Fluhr
Introduction The stratum corneum is viewed currently as a layer of protein-enriched corneocytes embedded in a lipidenriched, intercellular matrix [1], the so-called bricks and mortar model. The ‘‘bricks’’ are corneocytes surrounded by a cornified cell envelope made up of proteins, mainly loricrin, filaggrin, and involucrin, and covalently bound to the hydroxyceramide molecules of a lipid envelope. These ‘‘bricks’’ are embedded in a ‘‘mortar’’ of lipid bilayers [2–4]. The so-called mortar contains a variety of intercellular lipids including, ceramides, free sterols and sterolesters, cholesterolsulfate, and free fatty acids. The stratum corneum continually renews itself, and there is a steady state between the proliferation and differentiation process of keratinocytes and desquamation of corneocytes. Two important forces are responsible for the adherence of corneocytes and build the functional barrier of the skin: the corneodesmosomes as a morphomechanical force and the intercellular lipids as a functional force. Entering the process of differentiation, keratinosomes containing lamellar structured lipid bilayers reach to the apical cell pole from the center of the cytosol in the stratum spinosum and are extruded at the border between the stratum granulosum and stratum corneum. In the intercellular space, lipids form bi- and multilamellar structures, adhering to the corneocytes [1, 2]. Apart from an intact barrier function, the water content of the epidermis depends on the so-called natural moisturizing factors (NMF). These are amino acids, lactic acid, pyrrolidone carboxylic acid, and urea which are released after the breakdown of filaggrin in the mid-portion of the stratum corneum corneocytes, exhibiting an osmotic force and thus binding water. The effect of age on the thickness of skin strata is one of the more controversial topics among dermatological researchers. Comparing measures of skin layer thickness between individuals (and among studies) is especially challenging due to the significant variation in the measurements between individuals and between sites within each individual. Light and electron microscopic studies
have provided important evidence for morphological changes in skin strata with age, even though there is a general agreement that skin thickness (in terms of epidermis, dermis, and also stratum corneum) decreases with age (> Fig. 36.1).
Changes in Stratum Corneum with Age There have been few attempts to measure the rate of corneocyte loss and desquamation in relation to the aging process. This is odd because desquamation is a very important process. Corneocyte size and renewal (or turnover) depend not only on the rate of input into the system (epidermopoiesis), but also on the rate at which cells are lost (desquamation). The epidermis shows a linear decrease in thickness with age, both in absolute terms and in cell number. The reduction in epidermal population size suggests that there may also be a decrease in the rate of production of epidermal cells, and the apparent lengthening of the stratum corneum renewal time seems to confirm it. In addition, there is some evidence that the rate of reepithelization of wounds decreases with age. Using tritiated thymidine and an autoradiographic labeling method, Kligman [5] reported a reduced value for an elderly cohort compared to a younger group; in a study comparing the effects of aging between sun-exposed and non-exposed sites, this has not been detected [6]. A more sensitive but complicated assay using the FACS fluorescent assay demonstrated an age-related decrease in the DNA synthesis and therefore in a longer cell cycle through the stratum corneum [7]. Stratum corneum cell turnover and replacement time have been evaluated using also the dansyl chloride staining technique. Dansyl chloride is a fluorescent dye, which penetrates the full thickness of the stratum corneum, and when applied topically to the skin in vivo, becomes florescent under Wood’s light [8]. The time the fluorescence takes to disappear corresponds to the turnover cycle of the stratum corneum; these studies have shown a progressive increase in the turnover time of the stratum corneum
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_36, # Springer-Verlag Berlin Heidelberg 2010
372
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Corneocyte Size and Cell Renewal: Effects of Aging and Sex Hormones
. Figure 36.1 Correlation between thickness of the stratum corneum and age (Marks R [10])
. Figure 36.2 Corneocyte surface area and age. There is a significantly positive correlation (Plewig G [11])
associated with increasing age [9]. The lengthening of the turnover implies a reduction in the desquamation rate, but this is not as large as thought. The reason is the increase of corneocyte size during aging. Thus, there are fewer corneocytes in an old individual’s stratum corneum compared to a young one, per unit volume (> Fig. 36.2). Studies measuring the release of corneocytes from the skin also showed that there is a decrease of corneocyte loss at least when measured under these experimental conditions [10]. The evolution of corneocyte size during the aging process has been studied by several authors; there is a consensus that the size progressively increases with age, even though there are body site and seasonal variations (changes due to hormonal status will be discussed later in this document). The more investigated sites are the arm and the forearm, and data show a progressive increase of corneocyte size from birth to age (> Fig. 36.3) [11–14]. Some differences have been reported between sunexposed and non-exposed areas (> Fig. 36.4) [15] where in general UV irradiation increases epidermal turnover leading to smaller corneocytes compared to a similar photo-protected site. Indeed, seasonal variations in corneocyte size have been reported with smaller corneocytes in summer as a consequence of prolonged solar irradiation [16]. In a study on professional cyclists, it was found that the size of corneocytes from the area of the
arm protected by the shirt was ‘‘normal,’’ while in the adjacent exposed site the area of the cells was significantly smaller [17]. In conclusion there is a correlation and an inverse relationship between stratum corneum turnover and dimensions of corneocytes (> Fig. 36.5).
Influence of Sex Hormones The influences of sex hormones on morphologic and functional parameters of the epidermis are of increasing interest. The effects of hormones and aging on stratum corneum structure, function, and composition are not yet known in detail. Although age-dependent factors have been studied, few data are available concerning changes in perimenopausal women [18] with a significantly decreased sebum content of the forehead in menopausal women and higher stratum corneum hydration of the forehead in late menopausal women. Influences of female hormones on the composition of stratum corneum sphingolipids have been described, as well as the negative impact of age on the biosynthesis of sphingolipids [19]. With age a decline occurs in hormone levels, especially in sex hormones like estrogen, testosterone, dehydroepiandrosterone, and growth hormones [20, 21]. Hormone replacement therapy (HRT) leads to an increase in
Corneocyte Size and Cell Renewal: Effects of Aging and Sex Hormones
. Figure 36.3 Evolution of the corneocyte size versus age on the forearm. Data of different groups from (Plewig G) [11] (solid squares), 12 (solid circles), 13 (open circles), 14 (open squares)
36
collagen content [22]. Under basal conditions the physiologic functions of stratum corneum seems to remain unchanged with age. Under stressed conditions, however, aged skin is more susceptible to barrier disruption than younger skin, i.e. an aged epidermal permeability barrier shows decreased cohesion as well as delayed barrier repair with age under stress conditions [23, 24]. In a recent study [25], corneocyte size in pre- and postmenopausal women of the same age group (40–50 years) was investigated and compared to men of the same decade using a videomicroscopic technique: despite the close age range, the significantly smaller corneocytes in premenopausal women versus postmenopausal women or men are likely to be attributed to the different levels of female sex hormones (> Fig. 36.6). The detected differences support the hypothesis that sexual hormones have an impact on corneocyte surface area. Female sex hormone levels of premenopausal women are supposed to be higher than those of nonhormonal substituted postmenopausal women or men, and thus the smaller corneocyte surface area could be explained by the influence of female sex hormones. The barrier function and the stratum corneum hydration parameters are not involved in this mechanism as no correlation between these parameters and corneocyte surface area was detectable. In this study, no other major differences in barrier function or stratum
. Figure 36.4 Comparative evolution of corneocyte size at different body sites (From Corcuff P, Leveque JL [15])
373
374
36
Corneocyte Size and Cell Renewal: Effects of Aging and Sex Hormones
. Figure 36.5 Epidermal turnover and corneocyte size as influenced by age. Increasing size of corneocytes derives from slowing down of turnover related to the aging process
. Figure 36.6 Corneocyte size pre- and postmenopause as compared to men of the same age. Menopause induces a fast increase in corneocyte size comparable to men of the same age (Fluhr et al. [25])
corneum water-holding properties have been detected (maybe for the close chronological age range of the groups investigated), even though there some reports in the literature on the positive impact of hormone replacement therapy on cutaneous mechanical properties and water-holding capacity [26, 27]. Further investigation is necessary to study the physiology of perimenopausal skin especially under stress conditions. The role of hormonal replacement has been documented in a study [28] where menopausal women who had been treated for at least 5 or 10 years, the biophysical measurements were significantly higher for the parameters evaluating hydration and sebum secretion, which generally decrease after the menopause, associated with higher values for the yellow
intensity parameter and the skin relief parameters on the forehead. The skin relief parameters on the forehead were significantly higher in menopausal women since at least 5 years and taking HRT. This is one of the few studies that have demonstrated an effect of exposure to HRT on skin color assessed by colorimetry, and on skin relief with an increase of the roughness parameters on the forehead. An investigation assessed the effect of HRT on the skin, using high frequency diagnostic ultrasound combined with computerized image analysis. The study was a crosssectional observational study carried out on 84 women (comprising 34 HRT users, 25 postmenopausal controls, and 25 premenopausal controls). The time that volunteers had been taking for HRT varied from 6 months to 6 years. The skin was shown to be thicker in the HRT group than in the postmenopausal control group [29]. An additional study evaluating the severity of facial wrinkling by an eight-point photographic scale in a sample of Korean women, estimated the HRT exposition impact among 85 postmenopausal women, comprising 15 taking HRT. HRT was found to be associated with a lower risk for facial wrinkling in the postmenopausal women group [30]. These results support the subjective impression and the clinical evaluation concerning the impact of HRT on the development and the severity of some properties associated with skin aging after menopause.
Conclusion The aging process, associated with hormonal changes in women during menopause has a significant impact on the
Corneocyte Size and Cell Renewal: Effects of Aging and Sex Hormones
physiology of the skin and the stratum corneum. In particular, at this level, corneocytes are larger due to the slowing down of the metabolic processes and to the keratinocyte turnover; this can cause changes in the physical properties of the upper layers causing some ‘‘cosmetic’’ effects such as decreased brightness and reduction of transcutaneous penetration; cell renewal is slower, even though desquamation rate seems to be constant. Hydration of the stratum corneum seems not to change too much during aging, despite contradictory reports: probably this is related to an uneven distribution of the water profile on the skin surface, which can be investigated today by new imaging techniques [31].
Cross-references > Stratum > The
Corneum Cell Layers Stratum Corneum and Aging
References 1. Landmann L. The epidermal permeability barrier. Anat Embryol (Berl). 1988;178:1–10. 2. Swartzenruber DC, Wertz PW, Madison KC, et al. Evidence that the corneocyte has a chemically bound lipid envelope. J Invest Dermatol. 1987;88:709–713. 3. Wertz PW, Downing DT. Covalently bound o-hydroxyacylsphingosine in the stratum corneum. Biochim Biophys Acta. 1987;917:108–111. 4. Steven AC, Steinert PM. Protein composition of cornified cell envelopes of epidermal keratinocytes. J Cell Sci. 1994;107:693–700. 5. Kligman AM. Perspectives and problems in cutaneous gerontology. J Invest Dermatol. 1979;73:39–56. 6. Marks R, et al. The effects of phoageing and intrinsic ageing on epidermal structure and function. G Ital Chir Dermatol Oncol. 1987;2:252–263. 7. Marks R. The epidermal engine. A commentary on epidermopoiesis, desquamation and their interrelationships. J Cosmet Sci. 1986;8:135–144. 8. Jansen LH, Hojyo-Tomoko MT, Kligman AM. Improved fluorescence staining technique for estimating turnover of the human stratum corneum. Br J Dermatol. 1974;90:9–14. 9. Roberts D, Marks R. Determination of age variations in the rate of desquamation. A comparison of four techniques. J Invest Dermatol. 1979;74:13–16. 10. Marks R. Measurement of biological ageing in human epidermis. Br J Dermatol. 1981;104:627–633. 11. Plewig G. Regional differences in cell sizes in the human stratum corneum II. Effect of sex and age. J Invest Dermatol. 1970;54:19–23. 12. Marks R, Nicholls S, King CS. Studies on isolated cornocytes. Int J Cosmet Sci. 1981;3:251–258. 13. Grove GL, Lavker RM, Hoelzle E, Kligman AM. Use of noon intrusive tests to monitor age associated changes in human skin. J Soc Cosmet Chem. 1981;32:15–26.
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14. Leveque JL, Corcuff P, DeRigal J, Agache P. In vivo studies on the evolution of physical properties of the human skin with age. Int J Dermatol. 1984;23:322–329. 15. Corcuff P, Leveque JL. Corneocyte changes after acute UV irradiation and chronic solar exposure. Photodermatology. 1988;5:110–115. 16. Hermann S, Scheuber E, Plewig G. Exfoliative cytology: effects of seasons. In: Marks R, Plewing G (eds) Stratum Corneum. Beriln: Springer-Verlag, 1983, pp. 181–185. 17. Leveque JL, Porte G, DeRgal J, Corcuff P, Francois AM, Saint-Leger D. Influence of chronic sun exposure on some biophysical parameters of the human skin; an in vivo study. J Cutan Aging Cosmet Dermatol. 1988;1:123–127. 18. Ohta H, Makita K, Kawashima T, Kinoshita S, Takenouchi M, Nozawa S. Relationship between dermato-physiological changes and hormonal status in pre-, peri-, and postmenopausal women. Maturitas. 1998;30:55–62. 19. Denda M, Koyama J, Hori J, Horii I, Takahashi M, Hara M, Tagami H. Age- and sex-dependent change in stratum corneum sphingolipids. Arch Dermatol Res. 1993;285:415–417. 20. Tazuke S, Khaw KT, Barrett-Connor E. Exogenous estrogen and endogenous sex hormones. Medicine (Baltimore). 1992;71:44–51. 21. Roshan S, Nader S, Orlander P. Review: ageing and hormones. Eur J Clin Invest. 1999;29:210–213. 22. Sauerbronn AVD, Fonseca AM, Bagnoli VR, Saldiva PH, Pinotti JA. The effects of systemic hormonal replacement therapy on the skin of postmenopausal women. Int J Gynaecol Obstet. 2000;68:35–41. 23. Ghadially R, Brown BE, Sequeira-Martin SM, Feingold KR, Elias PM. The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model. J Clin Invest. 1995;95:2281–2290. 24. Reed JT, Ghadially R, Elias PM. Skin type, but neither race nor gender influence epidermal permeability barrier function. Arch Dermatol. 1995;131:1134–1138. 25. Fluhr JW, Pelosi A, Lazzerini S, Dikstein S, Berardesca E. Differences in corneocyte surface area in pre- and post-menopausal women: assessment with the noninvasive videomicroscopic imaging of corneocytes method (VIC) under basal conditions. Skin Pharmacol Appl Skin Physiol. 2001;14(Suppl 1):10–16. 26. Pierard-Franchimont C, Letawe C, Goffin V, Pierard GE. Skin waterholding capacity and transdermal estrogen therapy for menopause: a pilot study. Maturitas. 1995;22:151–154. 27. Pierard GE, Letawe C, Dowlati A, Pierard-Franchimont C. Effect of hormone replacement therapy for menopause on the mechanical properties of skin. J Am Geriatr Soc. 1995;43:662–665. 28. Guinot C, et al. Effect of hormonal replacement therapy on skin biophysical properties of menopausal women. Skin Res Technol. 2005;11:201–204. 29. Chen L, Dyson M, Rymer J, et al. The use of high frequency diagnostic ultrasound to investigate the effect of hormone replacement therapy on skin thickness. Skin Res Technol. 2001;7:95–97. 30. Youn CS, Kwon OS, Won CH, et al. Effect of pregnancy and menopause on facial wrinkling in women. Acta Dermatol Venereol. 2003;83:419–424. 31. Batisse D, Giron F, Leveque JL. Capacitance imaging of skin surface. Skin Res Technol. 2006;12:99–104.
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39 Cyanoacrylate Skin Surface Strippings Claudine Pie´rard-Franchimont . Jorge Arrese-Estrada . Pascale Quatresooz . Ge´rald E. Pie´rard
Introduction In its most widely appreciated context, the stratum corneum (SC) exerts a major barrier function extending to protection from ultraviolet light, microorganisms, oxidants, and other toxic xenobiotics. In addition, it protects from loss of water and electrolytes from the body. Despite limited metabolic activity, the SC is viewed as a highly specialized structure showing continuous renewal keeping ideally a steady state in its structure and thickness. However, it is structurally and biochemically heterogeneous. In addition, it acts as a unique sophisticated biosensor that signals the underlying epidermis to respond to a series of external stresses. On most body sites, the SC typically consists of 12–16 layers of flattened corneocytes. These cells are about 1 mm thick and have a mean area of approximately 1,000 mm2. However, the surface area depends on age, anatomical location, and conditions that influence the epidermal renewal such as chemical irritation and UV irradiation. In particular, the average corneocyte size increases with age. This is sometimes assumed to be related to the increased transit time within the SC. Each corneocyte contains a water-insoluble protein complex made predominantly of a highly organized keratin microfibrillar matrix. Such a structure is encapsulated in a protein- and lipid-enriched shell. This cornified cell envelope shows differences in maturation among corneocytes. Two distinct types of cornified cell envelopes were distinguished as ‘‘fragile’’ and ‘‘rigid,’’ or ‘‘immature’’ and ‘‘mature’’ [1, 2]. In some instances, the SC homeostasis is altered. Indeed, the SC is the repository of many biological events that occurred below it in previous days. The SC structure is further altered by diverse and repeated external threats. The genetic background, nutritional status, and some physical agents, as well as drugs, cosmetics, toiletries, and other chemical xenobiotics represent other major modulators of the SC structure. Knowledge about fine SC structure is crucial in many aspects of the dermocosmetic science, particularly when dealing with age-related xerosis and effects of surfactants, emollients, and squamolytic agents [3].
Critical Factors for Clinical Practicability of CSSS Cyanoacrylate skin surface stripping (CSSS) is a timehonored method [4]. After its clever discovery, it was soon applied for diagnostic purposes in dermatology. The CSSS method consists of depositing a drop of cyanoacrylate liquid adhesive onto a supple transparent sheet of terephthalate polyethylene, 175 mm thick, cut to the size of a conventional coverslip (1.5 6 cm). The material (3S-biokit, C + K electronic, Germany) is pressed firmly on the lesion. After 15–30 s, a sheet of SC of uniform thickness can be conveniently harvested (> Fig. 39.1). As the adhesion mechanism of cyanoacrylate relies on a chemical reaction, the depth of the removed SC is determined by the depth of penetration of the adhesive before it hardens. The cleavage level is exclusively located inside the SC. Oozing and eroded lesions are not adequately studied using CSSS. The sampling procedure is often painless and bloodless. Anesthesia and antiseptic procedures are unnecessary. The cost is minimal. The following laboratory procedure is simple and not timeconsuming. CSSS are conveniently harvested from any part of the body, with two main provisos. On the one hand, sampling from a hairy area is painful because of pulling out hairs. In addition, the CSSS quality may be inadequate owing to the poor contact with the SC. It is, therefore, advisable to shave these areas before sampling a CSSS. On the other hand, intercorneocyte cohesion on the palms and soles is frequently stronger than the cyanoacrylate bond, thus impairing the collection of an unbroken sheet of corneocytes. However, a CSSS sampling from these sites is possible in certain physiopathological conditions associated with a compromised texture and cohesion of the SC.
Overall Microscopic Aspect of Normal Skin on CSSS CSSS of normal skin reveals a regular network of highpeaked crests related to the skin surface hollow depressions corresponding to the so-called first- and second-order lines.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_39, # Springer-Verlag Berlin Heidelberg 2010
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Their pattern is typical for specific parts of the body and is subject to variations with age [5]. The primary lines of the skin surface correspond to grooves in the latticework papillary relief at the dermo–epidermal junction [3]. In young individuals, regular intersections of primary and secondary lines delimit regularly shaped polyhedral plateaus (> Fig. 39.2). With aging, this network progressively loses its configuration, being preferentially oriented along the skin tension lines. The process ends with the disappearance of the shallow wrinkles [6]. It is, therefore, possible to indirectly assess the texture of the superficial dermis on CSSS. Accordingly, dermal aging, corticosteroidinduced atrophy, sclerosis, striae distensae, scars, and many other changes in the connective tissue are readily visible in a noninvasive way using CSSS. This morphological assessment of the skin microrelief is conveniently quantified by computerized image analysis using any regular profilometric method [3]. . Figure 39.1 CSSS sampling
. Figure 39.2 CSSS: regular crisscross pattern of primary and secondary order lines of the skin surface
Velus hairs are commonly captured within the CSSS. In addition, CSSS collects follicular casts corresponding to the horny material present at the opening of the pilosebaceous follicles near the skin surface [7]. It is therefore possible to assess the density of the follicles per unit surface area, and to observe the presence of follicular hyperkeratosis (kerosis) as well as comedones, trichostasis spinulosa, intrafollicular bacteria, and mites [3, 5, 7–9].
Cytological Aspects of Normal Skin on CSSS Cytological characteristics of corneocytes are hardly visible on CSSS unless histological dyes are used [5]. A number of stains are suitable. The most useful and simplest one is a mixture of toluidine blue and basic fuschin in 30% ethanol. Normal skin shows a regular cohesive pattern of adjacent anucleated corneocytes. Their boundaries are clearly identified as a thin polyhedral rim (> Fig. 39.3). Parakeratotic cells are rare and dispersed singly on normal skin. They are recognized by the presence of a nucleus central to the polyhedral cell. Saprophytic microorganisms are present at the skin surface forming the biocene of resident bacteria. Thus, most of them are encased within the cyanoacrylate bond during sampling, and they are not accessible to the staining procedure. As a result, only a portion of the surface microflora is seen on CSSS [5]. By contrast, microorganisms present inside the follicular casts are collected distinctly from the skin surface biocene by scraping out these horny spiky structures appending to the CSSS. Viability of the intrafollicular bacteria can be assessed using flow cytometry [10].
. Figure 39.3 CSSS: regular corneocyte paving
Cyanoacrylate Skin Surface Strippings
Corneomelametry Melanin is present in normal corneocytes of phototype V and VI individuals. The dusty load can be specifically revealed using argentaffin-staining procedures. The relative darkness of these CSSS can be assessed using corneomelametry [11, 12]. This method consists of measuring the reduction of light transmission through the CSSS using a photomicroscope equipped with an internal photodensitometer device. On a cytological viewpoint, it is important to distinguish melanin-laden anucleated corneocytes from neoplastic dendritic melanocytes after their migration into the SC covering a malignant melanoma.
Diagnostic CSSS in Inflammatory Conditions Obviously the diagnostic indications for CSSS only apply to disorders characterized by changes taking place in the SC. Many common dermatoses are conveniently diagnosed using CSSS [3, 5, 13]. Straightforward diagnoses can be established in superficial, infectious, and parasitic skin diseases. Morphological examination, possibly combined with fungal cultures, can be carried out to identify these dermatoses. By essence, infectious agents that are made visible on CSSS are not those adhering on top of the skin surface (see above), but rather those invading the SC. Fungi, including yeasts and dermatophytes, exhibit typical morphology (> Fig. 39.4), forming clusters or a network of globular or filamentous structures. In the group of parasitic disorders, scabies may pose problem at the time of sampling. In fact, this diagnosis . Figure 39.4 CSSS of a dermatophytosis: fungal hyphae are clearly identified
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can be established only if the mite, its eggs, or its dejecta are present in the sample. Duplicate CSSS should therefore be sampled from a typical scabies burrow. The first one removes the roof of the burrow and the second one may collect the parasite. Any sample taken outside such lesion, for instance, from nonspecific prurigo, will be unhelpful because the diagnosis will only suggest the presence of a spongiotic dermatitis [5, 14]. Demodex mites are conveniently recognized [5, 14] and highlighted in the follicular casts using the Fite stain. Noninfectious erythemato-squamous disorders conveniently assessed using CSSS include spongiotic and parakeratotic dermatoses and xeroses [3, 5, 14]. Spongiotic dermatitides represent superficial inflammatory reactions responsible for spongiosis, microvesiculation, and serosity leakage inside the SC. Contact dermatitis, atopic dermatitis, and pityriasis rosea are examples that belong to the spongiotic group. Parakeratotic dermatoses encompass id reactions, chronic eczema, and stable psoriasis. The parakeratotic cells are clustered in sheets or thicker bulks (> Fig. 39.5). Seborrheic dermatitis also comes within this parakeratotic category particularly in cases when Malassezia yeasts are rare. In active psoriasis, clusters of neutrophils are found in the center of parakeratotsic foci [14].
Diagnostic CSSS in Cutaneous Neoplasms Some epithelial neoplasms display typical aspects on CSSS. Seborrhoeic keratoses show spotty lenticular foci of soft hyperkeratosis. Widening of shallow furrows with hyperkeratosis is often present [5]. Samples of actinic
. Figure 39.5 CSSS: sheet of parakeratoic cells
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keratosis often exhibit irregular thickness with interfollicular parakeratosis and xerosis. The perifollicular rim is, by contrast, featureless. Basal cell and squamous cell carcinomas do not exhibit specific features or suggestive clues on CSSS. Actinic porokeratosis is revealed by the rim of cornoid lamellation and loss of the normal microrelief inside the lesion [5]. Verrucous surfaces overlying melanocytic nevi and dermatofibromas are less pathognomonic, but sharp circumscription with normal surrounding skin and uniformity of the changes in the texture of the SC usually are seen in such benign neoplasms. In melanocytic neoplasms, melanin is found inside corneocytes and eventually in atypical melanocytes. Melanin restricted only inside corneocytes is a feature of benign neoplasms such as lentigines and melanocytic nevi. By contrast, the presence of atypical melanocytes inside the SC is strongly suggestive of malignant melanoma, but also, in rare instances, of a benign melanoacanthoma [5, 13–15]. Thus, CSSS proves to be sensitive and specific for distinguishing malignant melanoma from benign melanocytic tumors such as common melanocytic nevi, dysplastic nevi, or pigmented seborrheic keratoses [13]. For research purposes, karyometry of neoplastic melanocytes is conveniently performed on CSSS [15].
CSSS Assessment of Disease Severity and Therapeutic Activity Disease severity and therapeutic improvement are possibly assessed noninvasively on CSSS exhibiting specific features in the SC. An example is given by xeroses, which correspond to various forms of predominantly orthokeratotic hyperkeratosis [3]. This condition encompasses what is commonly referred to as sensitive skin or dry skin, but this appearance is also found to a more severe degree in ichthyoses [3, 5, 14]. Several types and grades of orthokeratotic hyperkeratosis are distinguished on CSSS [3, 5, 14]. Type 0 is the absence of hyperkeratosis, except for some discrete focal accumulation of corneocytes in the primary order lines of the skin. Type 1a corresponds to a continuous linear hyperkeratosis of the primary lines. Type 1b is characterized by hyperkeratosis predominant at the site of adnexal openings either at hair follicles or at acrosyringia. Type 2 corresponds to focal hyperkeratosis of the skin surface plateaus covering less than 30% of the surface of the sampling. Type 3 resembles type 2, but with a xerotic area over 30% of the CSSS. Type 4 is defined by a homogeneous and diffuse hyperkeratosis with persistence of the trace of primary order lines. Type 5a resembles Type 4, but with
loss of recognizable primary lines. Type 5b corresponds to the most heterogeneous and diffuse hyperkeratosis with loss of or marked remodeling of the primary line network.
Corneofungimetry In superficial dermatomycoses, fungal cells are readily visible on CSSS. In experimental settings, some assessments of disease severity and therapeutic activity on dermatomycoses can be performed on CSSS using computerized image analysis. In an in vitro procedure, fungi are conveniently cultured using corneocytes [16], and particularly CSSS as growth substrates [17, 18]. Quantifications of the restricted fungal growth after the application of antifungals in experimental dermatomycosis are conveniently performed using corneofungimetry [17, 19–21]. The oral or topical antifungals are administered to healthy volunteers for a given period of time (usually a couple of days). CSSS are sampled afterward. A controlled amount of fungal cells collected from a primary culture is deposited onto the CSSS supposedly impregnated by the antifungal test. After a given time (usually 7–10 days) of culture in a clean environment, the CSSS are stained for revealing fungi. Computerized image analysis is used to fine-tune the quantification of the mycelium growing on CSSS. The comparison with control untreated CSSS allows to derive the percentage of inhibition of the fungal growth. Corneofungimetry has several advantages over conventional in vitro evaluation of antifungals: (a) the treatment is applied in vivo in conditions normally encountered by patients, (b) the initial fungal load is controlled, (c) the growth medium is only composed of keratinocytes without any artificial compounds, and (d) any influence of keratinocytes including natural antimicrobial peptides is respected.
Comedometry Comedometry allows the computerized quantification of the number and size of follicular casts present on CSSS. This method finds application in the assessment of comedogenesis-related disorders and in their treatments [9, 10, 22]. Acne is the major indication. In vivo comedometry on human skin appears more relevant than animal models of comedogenesis. The sensitivity of the method is such that microcomedolysis is possibly objectivated by computerized image analysis after a few days or weeks of treatment.
Cyanoacrylate Skin Surface Strippings
Corneosurfametry and Corneoxenometry The interaction between the SC and various chemical xenobiotics is conveniently assessed on CSSS. Corneosurfametry (CSM) refers to the effects of surfactants and wash solutions [3, 23–25]; CSSS are harvested from healthy volunteers. A solution of the test product is sprayed on the CSSS which are placed in covered plastic trays. After a given time of incubation at controlled temperature, the samples are thoroughly rinsed in tap water, dried and stained for 3 min in a toluidine blue-basic fuschin solution. The samples are then copiously rinsed with water and dried prior to color determination using reflectance colorimetry. Indeed, surfactants remove lipids and denaturate corneocyte proteins, thus revealing sites available for staining deposition. A combined dotted and rimmed pattern is visible at the microscopic examination (> Fig. 39.6). Using quantitative reflectance colorimetry, mean luminancy (L*) and Chroma C* are calculated from measurements made at three sites on each sample placed on a white reference plate. It is known that mild surfactants with little effect on corneocytes give high L* values and low Chroma C* values. L* decreases and Chroma C* increases with the irritancy potential of the product. The differences between L* and Chroma C* values of each sample give colorimetric indices of mildness (CIM). The CSM index (CSMI) of the test product, corresponds to the difference in color between water-treated control . Figure 39.6 CSM bioassay: corneocyte alterations due to surfactants
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samples and those exposed to the test product. It is conveniently calculated according to the following formula: CSMI = [(DL*)2 + (DC*)2]0.5. Microwave CSM is a more rapid procedure [26]. CSSS are immersed in a flask containing the test surfactant solution. Samples are then placed in a microwave oven with a 500-mL water load. Microwave CSM is typically run at 750 W for 30 s. The next steps are identical to the standard CSM procedure. Responsive CSM is a variant of the method where skin has been pretreated before CSSS sampling [27]. The method is based on repeat subclinical injuries by surfactants monitored in a controlled forearm immersion test. At completion of the in vivo procedure, CSSS are harvested for a regular or microwave CSM bioassay using the same surfactant as in the preliminary in vivo procedure. Preconditioning the skin in this way increases CSM sensitivity to discriminate among mild surfactants [27]. Shielded CSM is used for testing skin protective products (SPP) [28]. SPP claiming for being barrier creams should be shields against noxious agents. In shielded CSM, the CSSS are first covered by the test SPP before performing regular CSM using a reference surfactant. Comparative screenings of SPP are conveniently performed using shielded CSM without exposing volunteers to hazards linked to in vivo testing. Animal CSM can be performed [29] in a way similar to human CSM. The method is available for safety testing of cleansing products specifically designed for some
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animal species. In addition, interspecies differences in surfactant reactivity of the skin to surfactants are conveniently assessed [29]. The corneoxenometry (CXM) bioassay is used for testing any chemical xenobiotic other than surfactants [28, 30, 31]. The basic procedure is similar to CSM and its variants. One main indication is found in the field of skin irritation while avoiding the in vivo hazards. Another indication concerns the comparative assessment of penetration enhancers commonly used in topical formulations [30].
Conclusion Besides conventional biopsies and cytology of exudates, imprints, and scrapings, CSSS provide useful information in the field of dermatopathology. This simple, rapid, cheap, and noninvasive method allows the clinician to avoid a conventional biopsy within the limits of welldefined indications. Less than 3 min are necessary between sampling and examination. There are evident features and subtle characteristics discernible in the structure of the SC that enable a diagnosis to be made in a variety of skin diseases. It is important to stress that no single criterion should usually be relied upon for a definitive diagnosis, but rather a constellation of clues should be sought. Quantifications are made possible on CSSS using computerassisted image analysis. A series of derived methods have been designed for investigative purposes. When performed under controlled procedures, the information appears reproducible and sensitive. In many instances, the procedures help to bypass animal testing and to avoid a number of hazards bound to in vivo human trials.
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5. Pie´rard-Franchimont C, Pie´rard GE. Assessment of aging and actinic damages by cyanoacrylate skin surface stripping. Am J Dermatopathol. 1987;9:500–509. 6. Quatresooz P, et al. The riddle of genuine skin microrelief and wrinkles. Int J Cosmet. 2006;28:389–395. 7. Uhoda E, et al. The conundrum of skin pores in dermocosmetology. Dermatology. 2005;210:3–7. 8. Pagnoni A, et al. Determination of density of follicles on various regions of the face by cyanoacrylate biopsy: correlation with sebum output. Br J Dermatol. 1994;131:862–865. 9. Letawe C, Boone M, Pie´rard GE. Digital image analysis of the effect of topically applied linoleic acid on acne microcomedones. Clin Exp Dermatol. 1998;23:56–58. 10. Pie´rard-Franchimont C, et al. Lymecycline and minocycline in inflammatory acne. A randomized, double-blind study on clinical and in vivo antibacterial efficacy. Skin pharmacol. Appl Skin Physiol. 2002;15:112–119. 11. Thirion L, Pie´rard-Franchimont C, Pie´rard GE. Whitening effect of a dermocosmetic formulation. A randomized double-blind controlled study on melasma. Int J Cosmet Sci. 2006;28:263–267. 12. Pie´rard-Franchimont C, et al. Analytic quantification of the bleaching effect of 4-hydroxyanisole-tretinoin combination on actinic lentigines. J Drugs Dermatol. 2008;7:873–878. 13. Pie´rard GE, et al. Cyanoacrylate skin surface strippings as an improved approach for distinguishing dysplastic nevi from malignant melanomas. J Cutan Pathol. 1989;16:180–182. 14. Pie´rard-Franchimont C, Pie´rard, GE. Skin surface stripping in diagnosing and monitoring inflamnatory, xerotic and neoplastic diseases. Pediatr Dermatol. 1985;2:180–184. 15. Pie´rard GE, et al. Karyometry of malignant melanoma cells present in skin strippings. Skin Res Technol. 1995;1:177–179. 16. Faergemann J. A new model for growth and filament production of Pityrosporum ovale (orbiculare) on human stratum corneum in vitro. J Invest Dermatol. 1989;92:117–119. 17. Rurangirwa A, Pie´rard-Franchimont C, Pie´rard GE. Culture of fungi on cyanoacrylate skin surface strippings: a quantitative bioassay for evaluating antifungal drugs. Clin Exp Dermatol. 1989;59:425–428. 18. Aljabre SHM, et al. Germination of Trichophyton mentagrophytes on human stratum corneum in vitro. J Med Vet Mycol. 1992; 30:145–152. 19. Pie´rard GE, Pie´rard-Franchimont C, Arrese Estrada J. Comparative study of the activity and lingering effect of topical antifungals. Skin Pharmacol. 1993;6:208–214. 20. Arrese JE, et al. Euclidean and fractal computer-assisted corneofungimetry. A comparison of 2% ketoconazole and 1% terbinafine topical formulations. Dermatology. 2002;204:222. 21. Pie´rard-Franchimont C, et al. Activity of the triazole antifungal R12663 as assessed by corneofungimetry. Skin Pharmacol Physiol. 2006;19:49–56. 22. Uhoda E, Pie´rard-Franchimont C, Pie´rard GE. Comedolysis by a lipohydroxyacid formulation in acne prone subjects. Eur J Dermatol. 2003;13:65–68. 23. Pie´rard GE, et al. Surfactant induced dermatitis. A comparison of corneosurfametry with predictive testing on human and reconstructed skin. J Am Acad Dermatol. 1995;33:462–469. 24. Henry F, et al. Regional differences in stratum corneum reactivity to surfactants: quantitative assessment using the corneosurfametry bioassay. Contact Dermatitis. 1997;37:271–275.
Cyanoacrylate Skin Surface Strippings 25. Xhauflaire-Uhoda E, et al. Skin capacitance imaging and corneosurfametry. A comparative assessment of the impact of surfactants on stratum corneum. Contact Dermatitis. 2006;54:249–253. 26. Goffin V, Pie´rard GE. Microwave corneosurfametry and the shortduration dansyl chloride extraction test for rating concentrated irritant surfactants. Dermatology. 2001;202:46–48. 27. Uhoda E, Goffin V, Pie´rard GE. Responsive corneosurfametry following in vivo preconditioning. Contact Dermatitis. 2003;49: 292–296. 28. Xhauflaire-Uhoda E, et al. Skin protection creams in medical settings: successful or evil? J Occup Med Toxicol. 2008;3:15–20.
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29. Goffin V, Fontaine J, Pie´rard GE, et al. Comparative surfactant reactivity of canine and human stratum corneum. A plea for the use of the corneosurfametry bioassay. Altern Lab Anim. 1999;27: 103–109. 30. Goffin V, et al. Penetration enhancers assessed by corneoxenometry. Skin Pharmacol Appl Skin Physiol. 2000;13:280–284. 31. Xhauflaire-UHoda E, Pie´rard-Franchimont C, Pie´rard GE. Effect of various concentrations of glycolic acid at the corneoxenometry and collaxenometry bioassays. J Cosmet Dermatol. 2008;7:198–294.
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Physiology
4 Degenerative Changes in Aging Skin Miranda A. Farage . Kenneth W. Miller . Howard I. Maibach
Introduction Over the last 2 centuries, medical progress has dramatically extended the human lifespan, more than doubling life expectancy across the world. Average global life expectancy has risen from about 25 (for both sexes) to 65 for men and 70 for women [1]. Women, whose average life expectancies exceed those of men, can now expect to spend more than one-third of their lifetimes in post menopause [2]. More than 40 million postmenopausal women live in the USA today, comprising 17% of the total population [3]. Although the human skin is incredibly durable, like all other organ systems it is affected by aging [3–5]. A sophisticated and dynamic organ comprising 17% of the body’s weight, the skin primarily acts as the barrier between the internal environment and the world outside. Yet it performs numerous functions beyond simply acting as a barrier [6]: homeostatic regulation, prevention of percutaneous loss of fluid, electrolytes, and proteins; temperature maintenance; sensory perception; and immune surveillance [7]. Aging involves both intrinsic and extrinsic processes occurring in parallel [8]. Intrinsic aging proceeds at different rates in all organisms at a genetically determined pace. It is caused primarily by the build-up of reactive oxygen species (ROS) as a by-product of cellular metabolism and by ROS-induced damage to critical cellular components like membranes, enzymes, and DNA. Skin cells become increasingly senescent as they age: [8] the rate of cell proliferation in the epidermis drops, which contributes to deterioration of skin structure and function [9]. Extrinsic aging is accelerated aging that is superimposed on intrinsic effects of age that result from environmental insults to the skin, often controllable exposures such as solar radiation [10]. As a human being ages, the skin thins, dries, wrinkles, and becomes unevenly pigmented [11]. A loss of subcutaneous fat, as well as underlying bone and cartilage, manifests as sagging skin and fallen nasal tips [12]. Skin complaints by older adults, particularly women, are largely esthetic – plastic surgery has become the fastest growing medical specialty [11] – but aging of the skin also can
produce significant morbidity. In fact, most people over 65 have at least one skin disorder, and many have two or more [13]. Chronic dryness and itching are particularly prevalent; in one study of healthy Japanese over 60 years of age, 95% suffered dry skin at least part of the year [14]. Irritant contact dermatitis associated with incontinence also rises among older adults [15, 16]. Various inflammatory, infectious, and vascular disorders become more common [17]. The prevalence of cutaneous malignancy also rises with age [17, 18]. Distinguishing the precludable aspects of cutaneous aging (primarily hormonal and lifestyle influences) from the inexorable (primarily intrinsic aging) is essential to preventing and treating the ailments of the aging skin. As the population ages, medical care of older skin must shift in focus from cosmetic improvements to reducing morbidity and mortality from dermatological disorders. This will improve the quality of life for the growing population of elderly adults [19].
Structure and Function of Normal Skin The skin is composed of three layers: epidermis, dermis, and hypodermis (> Fig. 4.1).
Epidermis The outer layer of the skin, the epidermis, contains primarily keratinocytes with smaller populations of melanocytes and immune cells (Langerhans cells) [19]. Epidermal thickness, which varies according to anatomic site and individual, averages from 50 to 100 mm [20]. The epidermis is a dynamic system whose structure and metabolism fulfill two main functions: to protect the skin from external insult and to maintain hydration of internal tissues [21]. Both functions are accomplished primarily by the stratum corneum, the outermost layer of the epidermis [22]. Epidermal keratinocytes originate in a single layer of cells at the basement membrane (the layer between the dermis and the epidermis). Cells produced at this layer move upwards; as they ascend, they
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_4, # Springer-Verlag Berlin Heidelberg 2010
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Degenerative Changes in Aging Skin
. Figure 4.1 Normal skin structure showing layers of epidermis, dermis and hypodermis
produce the definitive skin-cell protein, keratin, as well as a variety of lipids. As the keratinocytes continue to move upward toward the skin surface, they change shape as they mature. The stratum corneum (SC), the surface layer of skin, is composed of the flattened cell bodies of dead keratinocytes, now called corneocytes [23]. The SC averages 15 layers over most of the body, but ranges from as little as 3 layers in the very thin skin under the eye [29] to more than 50 layers on the palms and soles of the feet [24]. A dynamic and metabolically interactive tissue [22], the SC, comprises about 60% structural proteins, 20% water, and 20% lipids [19, 25]. The corneocytes of the SC are covered by a highly cross-linked and cornified envelope. The extracellular lipid lamellae consist of ceramides, long-chain free fatty acids, and cholesterol. The ceramides strongly adhere to the cornified envelope of the corneocytes, yielding a barrier membrane which, in healthy adults, maintains the water content of the viable portion of the epidermis at about 70% [26]. The strength of the water barrier also depends on its specific lipid composition [22] and relative
proportions of cholesterol, ceramides, and free fatty acids [19, 22]. These intercellular lipids, as well as sebum, natural moisturizing factor (NMF), organic acids and inorganic ions, impart the water-holding capacity of the SC [23]. Several minor components also contribute to maintaining skin hydration. Hyaluronic acid, a major waterbinding component of the dermis, has been recently shown to play a role in the barrier function and hydration of the SC [27]. Glycerol, which acts as an endogenous humectant, recently has been identified as another component of the SC [28]. In addition, a water-transporting protein named aquaporin-3, expressed from the basal layer up to one cell layer below the SC, acts to facilitate the movement of water between the basal layer of the epidermis and the SC in order to maintain a constant level of hydration in the viable epidermis [29]. The water content of the SC (about 20%) contrasts dramatically with that of the epidermis (about 70%), a sharp drop observable at the juncture between the stratum granulosum (SG) and the SC [26]. Tight junction structures, recently identified at the corneo-epidermal
Degenerative Changes in Aging Skin
junction, are protein aggregates that both control paracellular permeability and act as another way to prevent water in the epidermis from escaping into the stratum corneum [30]. When the barrier function and water water-retaining capacity of the SC is compromised [19], pathologic skin dryness can develop, at which point the stratum corneum becomes less flexible and begins to crack or fissure [21]. Skin is considered clinically dry when moisture content of the stratum corneum falls below 10%. Skin dehydration and cracking may facilitate entry of pathogenic microbes [19].
Dermis The dermis is a dense and irregular layer of connective tissue, 2 to 3 mm thick, that comprises most of the skin thickness (> Fig. 4.1) [2]. Dermal connective tissue contains elastin and collagen; collagen fibers contribute most of the mass of the skin and the bulk of its tensile strength [2]; elastin fibers provide elasticity and resilience [2]. The dermis also contains much of the skin’s vasculature, its nerve fibers and sensory receptors, and its
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primary water-holding components, i.e. hyaluronic acid (responsible for normal turgor of dermis because of its extraordinary water-holding capacity) and supportive glycosaminoglycans [2]. The dermis also serves as underpinning to the epidermis and binds it to the hypodermis [31].
Hypodermis The hypodermis is a layer of loose connective tissue below the dermis (> Fig. 4.1). It contains the larger blood vessels of the skin, subcutaneous fat (for energy storage and cushioning), and areolar connective tissue. The hypodermis provides cushioning, insulation, and thermoregulation, and it stabilizes the skin by connecting the dermis to the internal organs (> Fig. 4.1) [32].
Structural Changes in Aged Skin Changes in the thickness and other characteristics of the epidermis and dermis as skin ages are detailed below (> Fig 4.2 and > Table 4.1).
. Figure 4.2 Differences in skin structure between young and aged skin. With permission from Informa HealthCare- MA Farage, KW Miller, P Elsner and HI Maibach. 2007. Structural Characteristics of the Aging Skin: A Review. Journal of Cutaneous and Ocular Toxicology 26:343–357
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Degenerative Changes in Aging Skin
. Table 4.1 Changes in the structure of aged skin Observed effect of aging Lower lipid content Epidermis
Dermis
Hypodermis Appendages
Reference [77]
Dermal–epidermal junction flattens
[55]
Number of enzymatically active melanocytes decreases by 8% to 20% per decade
[20]
Number of Langerhans cells decreases
[41]
Capacity for re-epithelization diminishes
[78]
Number of pores increases
[79]
Thickness reduced (atrophy)
[36]
Vascularity and cellularity decrease
[12]
Collagen synthesis decreases
[44]
Pacinian and Meissner’s corpuscles degenerate
[44]
Structure of sweat glands becomes distorted, number of functional sweat glands decreases
[47]
Elastic fibers degrade
[21]
Number of blood vessels decreases
[12]
Number of nerve endings reduced
[9]
Distribution of subcutaneous fat changes
[44]
Overall volume decreases
[12]
Hair loses normal pigments
[44]
Hair thins
[44]
Number of sweat glands decreases
[44]
Nail plates become abnormal
[44]
Sebum production reduced
[79]
Skin Thickness Skin thickness rises over the first 20 years of life; subsequently, even though the number of cell layers remains stable, [33] adult skin thins progressively at a rate that accelerates with age [37]. This phenomenon occurs in all layers of the skin. The epidermis decreases in thickness with age [18]. The unexposed epidermis thins by up to 50% between the ages of 30 and 80 [34], but changes in epidermal thickness are most pronounced in exposed areas, such as the face, neck, upper part of the chest and the extensor surface of the hands and forearms [35]. Overall, epidermal thickness decreases at about 6.4% per decade [36, 37], decreasing faster in women than in men. Dermal thickness, [36] vascularity and cellularity also decrease with age. [12] The loss of dermal collagen and elastin makes up most of the reduction in total skin thickness in elderly adults: for example, in postmenopausal women, a decrease in skin thickness of 1.13% per year parallels a 2% decrease per year in collagen content [38].
Dermal thickness decreases at the same rate in both genders [21]. The hypodermis loses much of its fatty cushion with age. The basement membrane, a very small fraction of the total skin thickness, actually increases in thickness with age [39].
Changes in Composition of Aging Skin Epidermis As skin ages, epidermal cell numbers [40] and the epidermal turnover rate decrease [32, 41]. Characteristic changes occur in each of the cell types in the epidermis. Cells of the basal layer become less uniform in size, although average cellular size rises [42]. Keratinocytes change shape as skin ages, becoming shorter and fatter [40]; corneocytes become bigger due to decreased epidermal turnover [33, 43]. Enzymatically active melanocytes
Degenerative Changes in Aging Skin
decrease at a rate of 8% to 20% per decade, resulting in uneven pigmentation in elderly skin [44]. Langerhans cells, like other epidermal cells, display more heterogeneous appearance and function [45]. The number of Langerhan’s cells in the epidermis also decreases with age, leading to impairment of cutaneous immunity [44]. Langerhans cells that are produced have been observed to have fewer dendrites and therefore less antigen-trapping capability [45]. Although the number of sebaceous glands in the epidermis does not change, sebum production decreases [44]; the evolutionary and biologic significance of this remains unclear. The water content of aged skin, particularly that of the stratum corneum, is lower than that of younger skin [18, 19, 21]. Age-related changes in the amino acid composition [21] reduce the amount of cutaneous NMF, thereby decreasing the skin’s water- binding capacity [19]. The water content of the SC decreases progressively with age and eventually falls below the level necessary for effective desquamation; this causes corneocytes to pile up and adhere to the skin surface, which accounts for the roughness, scaliness, flaking that accompanies xerosis in aged skin. The integrity of the SC barrier is dependent on an orderly arrangement of critical lipids [3]. However, total lipid content of the aged skin decreases by as much as 65% [40]. Ceramide levels, particularly ceramide 1 linoleate [46] and ceramide 3 [47], are particularly depleted in older skin. Triglycerides are also reduced, as is the sterol ester fraction of stratum corneum lipids [18]. Although the levels of NMF in the SC are higher in aged skin than in younger (a consequence of the slower rate of epidermal turnover in older individuals [23]), amino acid levels are lower [28]. Corneocytes are fewer but much larger [28], with higher intercorneal cohesiveness [48]. Because permeability does not appear to be significantly increased in the skin of the aged individual, it has been generally assumed that barrier function does not alter significantly with aging [49]. Some differences in barrier function parameters, however, have been noted: Baseline transepidermal water loss (TEWL), a measure of the functional capacity of the stratum corneum to maintain the moisture content of the skin, however, is lower in older patients as compared to younger [18, 50], an observation believed to be due to the reduction of the water content of aged skin. (The elderly have less water to lose [50].) Recovery of baseline TEWL values after occlusion is also impaired in older skin [18]. In addition, it has been demonstrated that the permeability barrier of aged skin is also more vulnerable to disruption. In a study which used tape-stripping to effect loss of barrier integrity, adults over 80 required only 18
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stripping as compared to 31 strippings in young and middle-aged adults. (Tape-stripping is a common method of abrogating the SC by removing one layer of skin at a time by applying and then removing a strip of tape.) Recovery of barrier function in the aged subjects was also dramatically different [49]. Only 15% of those older than 80 had recovered barrier function at 24 h (as assessed by return to baseline TEWL), compared to 50% of the younger group [49]. Artificially-induced water gradients (such as produced by occlusion) were shown, in addition, to dissipate more slowly in older skin than in younger, again indicating reduced recovery capacity in aged skin [51]. The findings reveal that aging may have a profound impact on barrier integrity even though barrier function appears normal. A profound abrogation of functional capacity is exposed when the epidermal permeability barrier is under stress, and the barrier function is more easily disturbed and less able to recover. Interestingly, one study found that as skin dries as an inevitable aspect of intrinsic aging, TEWL and the water content of the stratum corneum drop in parallel, while in pathological conditions, TEWL increases even though stratum corneum water content stays low. In stripped skin both values increase, confirming a derangement of actual barrier function as skin ages [52]. The most widely observed structural change in aged skin is a flattening of the dermal–epidermal junction, which occurs as a result of the decreasing numbers and size of dermal papillae [53]. Histological studies reveal that the number of papillae per unit of area decreases dramatically [54], from an average of 40 papillae/mm2 in young skin, down to 14 papillae/mm2 in those aged over 65 [53]. The flattening of the dermal–epidermal junction, observed by about the sixth decade [36], creates a thinner epidermis primarily because of retraction of rete pegs [36], decreasing the thickness of the dermal– epidermal junction by 35% [32, 55]. As a consequence of the reduced interdigitation between dermis and epidermis and the flattened dermal– epidermal junction, the skin becomes less resistant to shearing forces and more vulnerable to insult [33]. Furthermore, flattening of the dermal–epidermal junction results in a smaller contiguous surface between the two layers and reduces communication between the dermis and epidermis; consequently the supply of nutrients and oxygen to the epidermis diminishes [32, 53]. This flattening also may limit basal cell proliferation and may affect percutaneous absorption [36]. The flattening of the dermal–epidermal junction may also contribute to wrinkle formation [33] by increasing the potential for dermal– epidermal separation [32, 53].
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Degenerative Changes in Aging Skin
Dermis
Hypodermis
The three major extracellular components of the dermis are collagen, elastin, and hyaluronic acid. All three are depleted in older skin. Collagen content decreases at about 2% per year [41], primarily because the production of matrix metalloproteinases, which degrade collagen, increases with age [56]. Degradation of dermal collagen by matrix metalloproteinases impairs the structural integrity of the dermis [57]. Mechanical tension or stress on dermal fibroblasts, created by a healthy collagen matrix, is critical for the maintenance of a proper balance between the synthesis of collagen and the synthesis of collagendegrading enzymes [58]. Fibroblast collapse, due to the accumulation of degraded collagen fibers that prohibit construction of a healthy collagen matrix, causes the ratio of collagen synthesis to collagen degradation to become deranged in a self-perpetuating cycle [57]. Aging is also associated with a decrease in collagen turnover (due to a decrease in fibroblasts and their collagen synthesis) [12] The relative proportions of collagen types are also disrupted over the lifespan. The proportion of Type I collagen to Type III collagen in young skin is approximately 6:1, a ratio which drops significantly over the lifespan as Type I collagen is selectively lost [59], although some increase in collagen Type III synthesis occurs as well [60]. In the aged dermis, collagen fibers become thicker and collagen bundles more disorganized than in younger skin [41]. Collagen cross-links stabilize, reducing elasticity in aged skin. Functional elastin also declines in the dermis with age, as elastin becomes calcified in aged skin and elastin fibers degrade [35]. Elastin turnover also declines [12]. The amount of glycosaminoglycans (GAGs), an important contributor to the structure and water-holding capacity of the dermis, declines with age [32, 53], as does the amount of hyaluronic acid produced by fibroblasts [32, 53] and the amount of interfibrillary ground substance, also a component of a healthy dermal matrix [61]. The loss of structural integrity of the dermis leads to increased rigidity, decreased torsion extensibility [32, 41] and diminished elasticity [2, 21], these properties eroding faster in women than in men [21], with a concomitant increase in vulnerability to shear force injuries [32, 41] The impact of these changes is dramatic: for example, when skin is mechanically depressed, recovery occurs in minutes in young skin, but takes over 24 h in skin of aged individuals [32, 41]. Perception of pressure and light touch also decrease in aged skin as pacinian and Meissner’s corpuscles degenerate. The number of mast cells and fibroblasts in the dermis also decreases [12].
The overall volume of subcutaneous fat typically diminishes with age, although the overall proportion of subcutaneous fat throughout the body increases until approximately age 70. Fat distribution changes as well; that in the face, hands, and feet decreases while a relative increase is observed in the thighs, waist, and abdomen. The physiological significance may be to increase thermoregulatory function by further insulating internal organs.
Physiological Changes Physiological changes in aged skin include changes in (i) biochemistry, (ii) neurosensory perception, (iii) permeability, (iv) vascularization, (v) response to injury, (vi) repair capacity, and (vii) increased incidence of some skin diseases as discussed below (> Table 4.2).
Biochemical Changes Vitamin D content of aged skin declines: synthesis of this compound slows because the dermis and epidermis lack its immediate biosynthetic precursor (7-dehydrocholesterol), which limits formation of the final product [41]. The surface pH of normal adult skin averages pH 5.5. This cutaneous acidity discourages bacterial colonization; it also contributes to the skin’s moisture barrier as amino acids, salts, and other substances in the acid mantle absorb water [54]. The pH of the skin is relatively constant from childhood to approximately age 70 [36], then rises significantly. This rise is especially pronounced in lower limbs, possibly due to impaired circulation [36].
Permeability The penetration and transit of permeants through the skin involves (i) absorption to the stratum corneum; (ii) diffusion through the stratum corneum, epidermis, and papillary dermis; and (iii) the removal by microcirculation [18]. The first two steps depend on the integrity and hydration of the stratum corneum, which in turn is a function of the level and composition of intracellular lipids [18]. The final step depends on the integrity of the microcirculation [18]. Heightened interest exists in transdermal administration of medications for long-term drug delivery in chronic disease, as this results in fewer side effects and promotes
Degenerative Changes in Aging Skin
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. Table 4.2 Changes in the function of aging skin Function Barrier function Sensory and pain perception Thermoregulation Response to injury
Permeability
Immune Function Miscellaneous
Change
Reference
Renewal time of stratum corneum increased by 50%
[41]
Baseline TEWL lower in elderly skin
[51]
Loss in sensitivity, especially after age 50
[33]
Increased itching
[80]
Decreased sweat production
[44]
Lower inflammatory response (erythema and edema)
[38]
Decreased wound healing
[41]
Reduced re-epithelization
[41]
Increased vulnerability to mechanical trauma
[41]
Decreased percutaneous absorption
[44]
Decreased sebum production
[41]
Decreased vascularization
[65]
Decreased chemical clearance
[41]
Decreased number of circulating thymus-derived lymphocytes
[33]
Decreased risk and intensity of delayed hypersensitivity reactions
[70]
Decreased Vitamin D production
[44]
Reduced elasticity
[2]
TEWL = transepidermal water loss
compliance. Consequently, data on percutaneous drug absorption in older adults have gained importance [62]. In general, older adults seem to absorb topical substances more slowly than younger subjects [63]. However, studies on percutaneous absorption in the aged have produced conflicting results. In people over 65, tetrachlorosalicylanilide was absorbed more slowly, but ammonium hydroxide was absorbed more rapidly, than in younger adults [33]. Increased permeability of aged skin to fluorescein and testosterone was observed in vitro [36, 41]. Absorption of radiolabeled testosterone was demonstrated to be three times that of younger subjects [29]. However, in a separate in vivo study, no difference between estradiol and testosterone absorption was observed in aged skin, while hydrocortisone and benzoic acid were both absorbed far less readily in aged skin as compared to younger [50]. These conflicting results may reflect compound- and body-site differences in the rates of percutaneous absorption [62]. Epidermal penetration of a substance is strongly associated with its hydrophobicity relative to the lipid content of the skin: consequently, hydrophobic compounds penetrate more readily in areas of the body areas that have high percentage of skin lipids. For example on the face, where the weight percentage of skin lipids is 12–15%,
hydrophobic compounds (lipophiles) penetrate more readily than hydrophilic ones, whereas on the soles of the feet, where the weight percentage of skin lipids is 1% to 2%, hydrophilic compounds penetrate more readily than hydrophobic ones [32, 50]. Using topically applied radiolabeled penetrants, excretion of lipophilic compounds, testosterone and estradiol, was compared to excretion of the more hydrophilic hydrocortisone and benzoic acids. Percutaneous absorption was quantified from urinary excretion profiles of radiolabel. No difference in percutaneous absorption of testosterone and estradiol was noted between younger and older skin, but absorption of both hydrocortisone and benzoic acid were nearly doubled in younger skin [62]. Because aged skin is drier and has a lower lipid content than younger skin, it may be less amenable to penetration by hydrophilic moieties [62] (> Table 4.3).
Vascularization and Thermoregulation In older skin, capillaries and small blood vessels regress and become more disorganized [33], blood vessel density diminishes [36], and a 30% reduction in the number of
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Degenerative Changes in Aging Skin
. Table 4.3 Percutaneous absorption of testosterone, estradiol, hydrocortisone, and benzoic acid in young and elderly people Cumulative % dose excreted in 5 days Compound
Molecular weighta
Log K (O/W)a
Testosterone
288.4
3.31
Estradiol
272.4
Hydrocortisone Benzoic acid
Aqueous solubility
Young
Elderly
Insoluble
13.2 3.0 n = 17
2.7
Almost insoluble
10.6 4.9 n=3
362.5
1.93
0.28 g/L
1.87 1.6 n = 15
15.2 8.4 n=7 9.0 5.6 n=6 0.86 0.5b n=9
122.1
1.87
3.4 g/L
42.6 16.5 n=6
23.1 7.0c n=9
Source: Ref. [62] a Values taken from the Merck Index; O/W = octanol–water partition b Significantly different from young control group at p < 0.01 c Significantly different from young control group, p < 0.05
venular cross sections per unit area of the skin surface occurs in non-exposed areas of the skin [33]. Capillaroscopy measurements using fluorescein angiography and native microscopy suggest a decrease in dermal papillary loops, which house the capillary network [36]. Although the pattern of blood flow through individual capillaries remains unchanged, [22] the maximum level of blood flow diminishes as functional capillary plexi are lost. A significant time delay in autonomic vasoconstriction in the aged (e.g., after postural changes, cold arm challenge, inspiratory gasp, body cooling) [18, 36] is well documented; this phenomenon is due primarily to declining function of the autonomic nervous system [18]. Eccrine sweating is markedly impaired with age. Spontaneous sweating in response to dry heat was 70% lower in healthy older subjects compared to young controls, due primarily to decreased output per gland [64]. Vascularity is also lost. Cross-sections of photodamaged skin reveal a 35% reduction in vascularity in the papillary dermis of aged skin [65], as well as reduced blood flow, depleted nutrient exchange, dysfunctional thermoregulation, reduced skin surface temperature, and increased skin pallor [66]. Facial skin temperatures were lower in aged subjects [38], and older people exhibited a wider temperature difference between groin and toes [33]. The elderly are predisposed to both hypothermia and heat stroke, as reduced eccrine sweating rates, lower vasodilation or vasoconstriction of dermal arterioles, and the loss of subcutaneous fat impair thermoregulation [64].
Irritant Response Inflammatory response to an exogenous agent declines in people over 70 years old [18, 32, 50]. The inflammatory response is slower and less intense, and some clinical signs of skin damage are absent [18, 38] (> Fig. 4.3). Diagnosis of common dermatological problems becomes difficult, and allergic sensitization tests may be meaningless [33]. Sunburn response also is attenuated and delayed [33]. Fewer inflammatory cells are seen in cantharidin blisters in older subjects [33]. The manifestation of skin irritation is blunted. Patch testing found less erythema, vesicles, pustules, and wheals in aged skin, as well as a decrease in TEWL [32, 55] in response to a range of skin irritants, including toilet soap [33], kerosene [33], dimethyl sulfoxide [DMSO], ethyl nicotinate, chloroform-methanol, lactic acid [18], chemicals which elicit inflammation by clearly different mechanisms [18]. In some cases, the response is also delayed. Analysis of changes in TEWL after sodium lauryl sulfate (SLS) application to the skin confirmed that in aged skin, the irritation reaction is slower and less frequent in postmenopausal then in premenopausal women [67]. Moreover, although blistering caused by ammonium hydroxide exposure is elicited more rapidly in older people, the time required to attain a full response is much longer than in younger ones [18]. The characteristics of the irritant response may be compound dependent in ways specific to older skin, as
Degenerative Changes in Aging Skin
. Figure 4.3 The inflammatory response is slower and less intense, and some clinical signs of skin damage are absent
chemical irritants induce their effects though different mechanisms. SLS as well as nonanoic acid disrupted keratinocyte metabolism and differentiation, while dithranol induced marked swelling of keratinocytes in the upper epidermis [32, 50]. In a study of croton oil, thymoquinone, and crotonaldehyde on older skin, decreased responsiveness was observed only to croton oil [18, 68].
Immune Response The immune response of aged skin is generally diminished. Numbers of Langerhans cells in the epidermis decrease by about 50% between the age of 25 and the age of 70 [41]. The total number of circulating lymphocytes decreases, as does the number of T-cells [32, 41] and B-cells [32, 41], both of which lose functional capacity with age [69]. Delayed hypersensitivity reactions decrease with age: numerous reports have demonstrated a decrease in the capacity for allergic response [32, 41, 70]. For example, healthy older subjects did not develop sensitivity to some known sensitizers and exhibited a lower frequency of positive reactions to standard test antigens compared to young adult controls [32, 41]. The frequency of IgE-mediated, positive prick tests to common allergens declined with age: peak reactivity was observed among people in their twenties, with 52% of subjects reacting to at least one test allergen; positive response rates dropped steadily with age, declining to 16% frequency among subjects older than 75 years [18]. Levels of circulating autoantibodies increase with age; this occurs in parallel with a decrease in useful antibodies as the aged individual’s existing immunity to specific allergens erodes [32, 46].
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Regenerative Capacity and Response to Injury In healthy skin, about one layer of corneoyctes desquamates every day, so that the whole stratum corneum replaces itself about every 2 weeks [23]. In contrast, elderly stratum corneum may take twice as long [71]. Repair of an impaired barrier requires the presence or the three main lipids in appropriate proportions [72] as well as stratum corneum turnover, both of which are suboptimal in older subjects. Injury repair diminishes with age. Wound-healing events begin later and proceed more slowly. For example, a wound area of 40 cm2, which in 20-year old subjects took 40 days to heal, required almost twice as long – 76 days – in those over 80 [32, 41]. The risk of post-operative wound reopening increased 600% in people in their mid-80s compared to those in their mid-thirties [32, 41]. The tensile strength of healing wounds decreased after the age of 70 [32, 41]. Repair processes like collagen remodeling, cellular proliferation, and wound metabolism are all delayed in the aged [32, 41]. The rate at which fibroblasts initiated migration in vitro following wound initiation was closely related to the age of the cell lines [32, 41, 73]. Barrier function requires twice as long to restore in the aged as compared to younger controls [74]; stratum corneum renewal times were much longer in the aged (about 30 days compared to 20 days in normal skin) [74]. Re-epithelialization of the stratum corneum after blistering is also diminished [75], being twice as long for people over 75 than for those aged 25 [32, 41]. The production of messenger RNA (mRNA) and IL-1 protein is also decreased in the aged, contributing to sluggish barrier recovery [75].
Neurosensory Perception Itching is reported more frequently by older adults. However, pain perception declines and pain perception is delayed after age 50 [33]. Consequently, the risk of tissue injury rises, as the most obvious warning signals – pain, erythema, and edema – appear more slowly [33]. This, coupled with longer wound repair times, results in higher morbidity in the aged.
Conclusion Humans now live to twice their reproductive age, an achievement that is unique [76]. Although many
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Degenerative Changes in Aging Skin
profound changes occur over a skin’s lifetime, the human integument remains relatively functional when protected from excessive environmenpal insult. However, the skin of older adults is compromised in many ways [18]. Structural changes lead to undesirable visible characteristics, as well as a decreased elasticity and resilience. Decreases in neurosensory capacity increase the risk of unrecognized injury. The intrinsic drying of the skin makes the skin itchy and increasingly uncomfortable. The decrease in the skin’s ability to repair itself slows wound repair and re-epithelization dramatically and increases the risk of surgical dehiscence. As the proportion of older adults in the industrialized world increases, caring for the problems of aged skin will improve the quality of life in those later years of life gained by medical advances.
Cross-references > Skin
Aging: A Brief Summary of Characteristic Changes
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Degenerative Changes in Aging Skin 39. Va´zquez F, Palacios S, Aleman˜ N, et al. Changes of the basement membrane and type iv collagen in human skin during aging. Maturitas. 1996;25:209–215. 40. Suter-Widmer J, Elsner P. Age and irritation. In: Agner T, Maibah H (eds) The Irritant Contact Dermatitis Syndrome. Boca Raton: CRC Press, 1996. 41. Fenske NA, Lober CW. Structural and functional changes of normal aging skin. J Am Acad Dermatol. 1986;15:571–585. 42. Bre´ge´ge`re F, Soroka Y, Bismuth J, et al. Cellular senescence in human keratinocytes: unchanged proteolytic capacity and increased protein load. Exp Gerontol. 2003;38:619–629. 43. Sauermann K, Jaspers S, Koop U, et al. Topically applied vitamin c increases the density of dermal papillae in aged human skin. BMC Dermatol. 2004;4:13. 44. Phillips T, Kanj L. Clinical manisfestations of skin aging. In: Squier C, Hill MW (eds) The Effect of Aging in Oral Mucosa and Skin. Boca Raton: CRC Press, 1994. 45. Wulf HC, Sandby-Møller J, Kobayasi T, et al. Skin aging and natural photoprotection. Micron. 2004;35:185–191. 46. Rogers J, Harding C, Mayo A, et al. Stratum corneum lipids: the effect of ageing and the seasons. Arch Dermatol Res. 1996;288:765–770. 47. Zettersten EM, Ghadially R, Feingold KR, et al. Optimal ratios of topical stratum corneum lipids improve barrier recovery in chronologically aged skin. J Am Acad Dermatol. 1997;37:403–408. 48. Long CC, Marks R. Stratum corneum changes in patients with senile pruritus. J Am Acad Dermatol. 1992;27:560–564. 49. Ghadially R, Brown BE, Sequeira-Martin SM, et al. The aged epidermal permeability barrier. structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model. J Clin Invest. 1995;95:2281–2290. 50. Ghadially R. Aging and the epidermal permeability barrier: implications for contact dermatitis. Am J Contact Dermatitis. 1998; 9:162–169. 51. Roskos KV, Guy RH. Assessment of skin barrier function using transepidermal water loss: effect of age. Pharm Res. 1989;6:949–953. 52. Berardesca E, Maibach HI. Transepidermal water loss and skin surface hydration in the non invasive assessment of stratum corneum function. Derm Beruf Umwelt. 1990;38:50–53. 53. Su¨del KM, Venzke K, Mielke H, et al. Novel aspects of intrinsic and extrinsic aging of human skin: beneficial effects of soy extract. Photochem Photobiol. 2005;81:581–587. 54. Fiers SA. Breaking the cycle: the etiology of incontinence dermatitis and evaluating and using skin care products. Ostomy Wound Manage. 1996;42:32–34, 36, 38–40, passim. 55. Neerken S, Lucassen GW, Bisschop MA, et al. Characterization of age-related effects in human skin: a comparative study that applies confocal laser scanning microscopy and optical coherence tomography. J Biomed Opt. 2004;9:274–281. 56. Ashcroft GS, Horan MA, Herrick SE, et al. Age-related differences in the temporal and spatial regulation of matrix metalloproteinases (mmps) in normal skin and acute cutaneous wounds of healthy humans. Cell Tissue Res. 1997;290:581–591. 57. Fisher GJ, Varani J, Voorhees JJ. Looking older: fibroblast collapse and therapeutic implications. Arch Dermatol. 2008;144:666–672. 58. Varani J, Dame MK, Rittie L, et al. Decreased collagen production in chronologically aged skin: roles of age-dependent alteration in fibroblast function and defective mechanical stimulation. Am J Pathol. 2006;168:1861–1868. 59. Oikarinen A. The aging of skin: chronoaging versus photoaging. Photodermatol Photoimmunol Photomed. 1990;7:3–4.
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60. Savvas M, Bishop J, Laurent G, et al. Type iii collagen content in the skin of postmenopausal women receiving oestradiol and testosterone implants. Br J Obstet Gynaecol. 1993;100:154–156. 61. Castelo-Branco C, Figueras F, Martı´nez de Osaba MJ, et al. Facial wrinkling in postmenopausal women. Effects of smoking status and hormone replacement therapy. Maturitas. 1998;29:75–86. 62. Roskos KV, Guy RH, Maibach HI. Percutaneous absorption in the aged. Dermatol Clin. 1986;4:455–465. 63. Kligman AM. The treatment of photoaged human skin by topical tretinoin. Drugs. 1989;38:1–8. 64. Ohta H, Makita K, Kawashima T, et al. Relationship between dermato-physiological changes and hormonal status in pre-, peri-, and postmenopausal women. Maturitas. 1998;30:55–62. 65. Gilchrest BA, Stoff JS, Soter NA. Chronologic aging alters the response to ultraviolet-induced inflammation in human skin. J Invest Dermatol. 1982;79:11–15. 66. Baumann L. Skin ageing and its treatment. J Pathol. 2007;211: 241–251. 67. Elsner P, Wilhelm D, Maibach HI. Sodium lauryl sulfateinduced irritant contact dermatitis in vulvar and forearm skin of premenopausal and postmenopausal women. J Am Acad Dermatol. 1990;23:648–652. 68. Coenraads PJ, Bleumink E, Nater JP. Susceptibility to primary irritants: age dependence and relation to contact allergic reactions. Contact Derm. 1975;1:377–381. 69. Szewczuk MR, Campbell RJ. Loss of immune competence with age may be due to auto-anti-idiotypic antibody regulation. Nature. 1980;286:164–166. 70. Robinson MK. Population differences in skin structure and physiology and the susceptibility to irritant and allergic contact dermatitis: implications for skin safety testing and risk assessment. Contact Derm. 1999;41:65–79. 71. Baker H, Blair CP. Cell replacement in the human stratum corneum in old age. Br J Dermatol. 1968;80:367–372. 72. Man MQM, Feingold KR, Thornfeldt CR, et al. Optimization of physiological lipid mixtures for barrier repair. J Invest Dermatol. 1996;106:1096–1101. 73. Muggleton-Harris AL, Reisert PS, Burghoff RL. In vitro characterization of response to stimulus (wounding) with regard to ageing in human skin fibroblasts. Mech Ageing Dev. 1982;19:37–43. 74. Grove GL, Kligman AM. Age-associated changes in human epidermal cell renewal. J Gerontol. 1983;38:137–142. 75. Barland CO, Zettersten E, Brown BS, et al. Imiquimod-induced interleukin-1 alpha stimulation improves barrier homeostasis in aged murine epidermis. J Invest Dermatol. 2004;122:330–336. 76. Naftolin F. Prevention during the menopause is critical for good health: skin studies support protracted hormone therapy. Fertil Steril. 2005;84:293–294, discussion 295. 77. Saint Le´ger D, Franc¸ois AM, Le´veˆque JL, et al. Age-associated changes in stratum corneum lipids and their relation to dryness. Dermatologica. 1988;177:159–164. 78. Holt DR, Kirk SJ, Regan MC, et al. Effect of age on wound healing in healthy human beings. Surgery. 1992;112:293–297; discussion 297–298. 79. Rawlings AV. Ethnic skin types: are there differences in skin structure and function? Int J Cosmet Sci. 2006;28:79–93. 80. Buckley C, Rustin MH. Management of irritable skin disorders in the elderly. Br J Hosp Med. 1990;44:24–26, 28, 30–32.
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Biomarkers
47 DNA Biomarkers in Aging Skin Kimberly G. Norman . Alex Eshaghian . James E. Sligh
Introduction Aging of the skin is the result of both the intrinsic chronological aging process and extrinsic damage caused by environmental factors. A major role of the skin is that of protection from external environmental factors. Ultraviolet radiation (UVR) is the most significant environmental insult to the skin. UVR comprises the spectrum of electromagnetic radiation between the wavelengths of 200 and 400 nm. UVR is subdivided into three categories, each of which has distinct biological effects: UVA (320– 400 nm), UVB (280–320 nm), and UVC (200–280 nm). The stratospheric ozone blocks the radiation whose wavelength is below 290 nm, effectively preventing the entire UVC spectrum and part of the UVB spectrum from reaching human skin. The UVR that does reach the human skin can cause molecular defects including DNA damage, lipid peroxidation, and protein cross-linking, which can lead to premature skin aging or photoaging. Photoaging is a term used to describe the clinical and histological features of chronically UV-exposed skin [1]. Photoaging occurs more frequently in people with fair skin and tends to be located in sun-exposed areas such as the head, neck, hands, and forearms. Sun-exposed areas of the skin exhibit characteristic features of aging in common with sun-protected, chronologically aged skin as well as with other chronologically aged tissues. However, certain features of sun-exposed skin are exclusive to these tissues. Hence, the term photoaging refers to the physiologic and pathological changes that occur specifically in aged tissue that has experienced chronic sun exposure over time. Clinical symptoms of photoaging include dry skin, formation of lentigines and nevi, hyperpigmentation, telangiectasia, leathery appearance, increased wrinkle formation, reduced recoil capacity, increased skin fragility, blister formation, and impaired wound healing ability [1, 2]. UVR also causes histologic changes in the skin including hyperkeratosis, thickening of the basement membrane, irregular melanocyte distribution, elastosis, dermal intercellular and perivascular edema, and perivascular infiltration [1]. Further changes include deposition of glycosaminoglycans, fragmented elastic fibers, and interstitial collagen.
Of the UV lights that reach the skin, UVB is mostly absorbed in the epidermis, whereas UVA penetrates through the epidermis and into the dermis. Therefore, UVB affects keratinocytes in the epidermis and UVA affects keratinocytes in the epidermis and fibroblasts in the dermis. UVB most commonly causes damage in the form of cyclobutane pyrimidine dimers (> Fig. 47.1). The characteristic hallmarks of UVB damage are C to T and CC to TT DNA changes. These occur in semiconservative DNA replication due to the A rule, which states that when DNA polymerase comes across un-interpretable changes, it inserts A residues by default. Thus, two A residues are inserted into DNA on strands opposite to cyclobutane-type cytosine-cytosine dimmers, leading to two TTresidues on the template strand. UVA, on the other hand, primarily causes DNA damage indirectly by the production of short-lived reactive oxygen species (ROS) such as singlet oxygen (O ), O2 , and H2O2 via endogenous photosensitizers. ROS leads to single-stranded DNA breaks, nucleotide changes, and DNA-protein cross-links. Potential sites of ROS-induced DNA damage are shown in > Fig. 47.2, and the formation of 8-hydroxyguanine, the most common altered base due to ROS, is shown in > Fig. 47.3. 8-Hydroxyguanine lesions are employed as a DNA marker of overall oxidative stress in the cell and UV damage in the skin. Singlet oxygen produced by UVA light has been shown to cause strand breaks in the mitochondrial DNA (mtDNA), which has resulted in mtDNA deletions [3]. mtDNA deletions are thought to be involved in the photoaging phenotype and serve as biomarkers of aging in the skin, based upon the observation that chronically UV-exposed skin with clinical signs of photoaging has a high frequency of mtDNA deletions as compared to UV-protected skin [4, 5].
Mitochondrial Implications in Photoaging Mitochondria have well-recognized roles both in the generation of cellular energy and as mediators of cellular events such as apoptosis. Mitochondria contain their own genome, a maternally inherited, circular, double-stranded DNA of 16,569 bp encoding 22 tRNAs, two rRNAs,
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_47, # Springer-Verlag Berlin Heidelberg 2010
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DNA Biomarkers in Aging Skin
. Figure 47.1 Cyclobutane-type pyrimidine dimer formation. The formation of cyclobutane-type pyrimidine dimers as a result of UVB is shown between thymidine (T) and cytosine (C) nucleotides
. Figure 47.2 Potential sites of oxidative DNA damage. The sites of potential DNA damage due to reactive oxygen species (ROS) are shown in red on the four deoxyribonucleotides. A: adenosine, G: guanosine, T: thymidine, and C: cytosine
. Figure 47.3 8-Hydroxyguanine formation. The formation of 8-hydroxyguanine is the most common DNA defect as a result of reactive oxygen species (ROS). 8-Hydroxyguanine can be formed when a hydroxide radical (OH ·) or H2O2 reacts with guanosine (G)
DNA Biomarkers in Aging Skin
and 13 polypeptides, all of which are subunits in the electron transport chain [6-8]. The remainder of the proteins that function in the mitochondrion are encoded in the nucleus and imported into the mitochondrion. Because the mitochondria contain multiple copies of the mtDNA and cells may contain thousands of mitochondria, newly acquired somatic mutations are heteroplasmic, or mixed with wild type mutations, in nature. However, replicative segregation may allow mutant mtDNA molecules in some cells to become prominent or even to become the exclusive mtDNA in the cell, a condition known as homoplasmy. The mitochondrion serves as the major site for the production of ATP through the process of oxidative phosphorylation (OXPHOS). ROS, natural by-products of this pathway, can damage lipids, proteins, and DNA [9, 10]. mtDNA has a high mutation rate due to its lack of histones, decreased capacity for repair, and its close proximity to the site of ROS formation [11]. Imbalances between oxidative stress and free radical scavenging enzymes have been suggested as the underlying causes of most of the mtDNA damage [12, 13]. There is emerging evidence for mtDNA changes in the complex processes of cellular aging and neoplasia [14–16]. The mitochondrial theories of aging hold as their basic principles that OXPHOS produces a main sources of cellular energy in the form of ATP and that there is an age-related decline in OXPHOS. The mtDNA may be particularly important in this energetic decline because its mutation rate is thought to be at least tenfold higher that that of the nuclear genome [17]. A multitude of factors are likely responsible for this increased mutation rate including the lack of protective histones and a less sophisticated system of proofreading than that present in the nucleus [18]. Also, mtDNA is exposed to higher concentrations of oxygen-free radicals as a consequence of their liberation from natural event occurring in the electron transport chain. Continued exposure of the mtDNA to oxidative damage results in an accumulation of somatic mtDNA mutations over time. These mutations may further decrease the efficiency of OXPHOS and increase the likelihood of additional oxygen-free radical production with further subsequent mtDNA damage. This cycle results in a progressive decline in the energy-generating capacity of the cell. Disease ensues when energy output falls below the minimum energetic threshold for normal tissue function. mtDNA changes have been associated with a variety of inherited and acquired human neurodegenerative disorders, myopathies, and endocrinopathies. Common characteristics of these clinical phenotypes are both delayed onset and age-related progression [16]. Age-related onset and a history of sun
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exposure are typical for acquired cases of non-melanoma skin cancer, which is the most common malignancy of older individuals and is one of the most frequently occurring health problems requiring surgical procedures in the elderly. Although there is substantial data correlating mtDNA changes with aging, in general, and with photoaging in the skin, there is no evidence of mtDNA mutations directly causing these phenotypes.
Mitochondrial DNA Mutations and Photoaging In contrast to other tissues, the skin is subject to both chronological aging and environmental insult in the form of UVR. The resulting genetic changes may lead to specific phenotypes of aging skin: photodamage and neoplasia. Changes in the mtDNA in the skin are well recognized in association with photoaging [19–21]. The most common mutation found in aging tissues is the 4,977 bp ‘‘common’’ deletion [22–28]. In autopsy specimens, the common deletion has been found primarily in sun-exposed areas and not in sun-protected areas [29]. Imbalances between oxidative stress and free radical scavenging enzymes have been suggested as the underlying causes of most of the mtDNA damage [12, 13]. The common deletion has been shown to be inducible both in vitro and in vivo in human skin and is thought to occur as a result of mtDNA damage mediated through singlet oxygen [3, 30]. Additionally, the 4,977 bp ‘‘common’’ deletion has been proposed to be a biomarker of photoaged skin because the level of heteroplasmy in the sun-exposed skin increases with age while such levels are not increased in sun-protected skin [29]. In a study by Ray et al. many mtDNA deletions in addition to the common deletion were identified in the epidermis of skin from older individuals and these deletions were strongly associated with UVR [4]. A report of 200 and 260 bp duplications in the non-coding D loop adds another class of mtDNA rearrangements that have been observed in aged human skin [31]. Additionally, an mtDNA deletion of 3,895 bp was identified as a quantitative marker for sunlight exposure in the human skin [32, 33], and the agingdependent T414G mutation within the control region of mtDNA was found to be accumulated in UVR-damaged skin [34]. It was also discovered that the T414G mutation, which may serve as both a marker for chronological aging and photoaging, was commonly identified within a 3,895 bp deleted mtDNA population. In a study in the authors’ lab to examine mtDNA mutations in photoaging, mtDNA from photodamaged skin was screened for the presence of deletions using long
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extension PCR. mtDNA deletions were found to be abundant in photoaged skin specimens from older patients and their number correlated with the patient age, supporting the use of mtDNA mutations as biomarkers of photoaging in the skin [35]. These mtDNA deletions were typically absent in the paired non-melanoma skin cancers. The observed DNA deletions from skin were often unreported (19 of 21 deletions), but usually shared structural features with mtDNA deletions reported in other tissues in that they generally contain short direct or indirect repeats at the breakpoints, and a single copy of the repeat is left behind in the deleted molecule. The structural similarities between the mtDNA deletions observed in the skin and those seen in tissues not exposed to UVR [36] imply a potential common factor in their generation or resolution. Some of the identified deletions were detected from the numerous skin samples, including 3,715 and 6,278 bp deletions. Interestingly, the frequency of these newly identified deletions approached that of the well-characterized 4,977 bp deletion. A consistently higher level of heteroplasmy of the 4,977 bp ‘‘common’’ deletion was found in the dermis as compared to the epidermis in split samples used, consistent with previous studies [29]. Furthermore, the number of novel deletions in photoaged skin not reported in other tissues suggests that skin may be more vulnerable to such mutations via direct exposure to UVR. The mechanism of formation of such mtDNA deletions has been proposed to be a slip mispairing of the repeats during replication [3, 13, 37, 38]. In order for such a mutation to occur, however, both breakpoints must be single stranded simultaneously, which does not normally occur. However, the sequences flanking the repeats or the sequences within the repeats may render the DNA susceptible to structural conformations allowing mispairing [39]. The findings show that most deletions identified contain such repeats, supporting the slip replication model. The novel 3,715 and 6,278 bp deletions contain 10 and 11 bp direct repeats, respectively, similar in size to that of the 4,977 bp ‘‘common’’ deletion (13 bp). Additionally, the novel 6,278 bp deletion contains a homopolymeric track of seven consecutive cytosine residues interrupted by a single adenosine residue within its repeated breakpoint sequence. Such sequences have been proposed to take on structural characteristics allowing for mispairing to occur [40]. These features may explain the reason why the frequencies of detection of the novel 3,715 and 6,278 bp deletions approached that of the 4,977 bp ‘‘common’’ deletion. It has been proposed that ROS, which are normally generated in response to UVR [41], play a role in the generation of the 4,977 bp
‘‘common’’ deletion [3]. Thus, it is not surprising that so many unreported mtDNA deletions were identified in photoaged skin given its role as a barrier to UVR. As mtDNA deletions accrue in photoaged skin, they may be useful as biomarkers of the combined effects of chronological aging and UV exposure. Additionally, specific mtDNA deletions may complement other deletions and the potential level of a single mtDNA deletion may plateau with time as it adversely affects the bioenergetic properties of the cell. Measurement of a panel of deletions may be a more useful assay of photodamage than heteroplasmy levels of any single deletion. Quantitative analysis of other deletions, in addition to the 4,977 bp ‘‘common’’ deletion may be useful for this purpose.
Conclusion While the 4,977 bp ‘‘common’’ deletion has been proposed to be a biomarker for photoaging, it may reflect only a small portion of total mtDNA damage. The 3,715, 6,278, and 3,895 bp deletions and T414G mutation may prove valuable as additional markers of photoaging in the skin. Furthermore, in this study, the photoaged skin contained an abundance of various deletions beyond the aforementioned mtDNA changes. Thus, the use of the 4,977 bp ‘‘common’’ deletion as a biomarker for photoaged skin may indeed be the tip of the iceberg.
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26. Ikebe S, Tanaka M, Ohno K, Sato W, Hattori K, Kondo T, Mizuno Y, Ozawa T. Increase of deleted mitochondrial DNA in the striatum in Parkinson’s disease and senescence. Biochem Biophys Res Commun. 1990;170:1044–1048. 27. Nagley P, Wei YH. Ageing and mammalian mitochondrial genetics. Trends Genet. 1998;14:513–517. 28. Sciacco M, Bonilla E, Schon EA, DiMauro S, Moraes CT. Distribution of wild-type and common deletion forms of mtDNA in normal and respiration-deficient muscle fibers from patients with mitochondrial myopathy. Hum Mol Genet. 1994;3:13–19. 29. Birch-Machin MA, Tindall M, Turner R, Haldane F, Rees JL. Mitochondrial DNA deletions in human skin reflect photo- rather than chronologic aging. J Invest Dermatol. 1998;110:149–152. 30. Berneburg M, Plettenberg H, Medve-Konig K, Pfahlberg A, GersBarlag H, Gefeller O, Krutmann J. Induction of the photoagingassociated mitochondrial common deletion in vivo in normal human skin. J Invest Dermatol. 2004;122:1277–1283. 31. Durham SE, Krishnan KJ, Betts J, Birch-Machin MA. Mitochondrial DNA damage in non-melanoma skin cancer. Br J Cancer. 2003;88:90–95. 32. Harbottle A, Birch-Machin MA. Real-time PCR analysis of a 3895 bp mitochondrial DNA deletion in nonmelanoma skin cancer and its use as a quantitative marker for sunlight exposure in human skin. Br J Cancer. 2006;94:1887–1893. 33. Krishnan KJ, Harbottle A, Birch-Machin MA. The use of a 3895 bp mitochondrial DNA deletion as a marker for sunlight exposure in human skin. J Invest Dermatol. 2004;123:1020–1024. 34. Birket MJ, Birch-Machin MA. Ultraviolet radiation exposure accelerates the accumulation of the aging-dependent T414G mitochondrial DNA mutation in human skin. Aging Cell. 2007;6:557–564. 35. Eshaghian A, Vleugels RA, Canter JA, McDonald MA, Stasko T, Sligh JE. Mitochondrial DNA deletions serve as biomarkers of aging in the skin, but are typically absent in nonmelanoma skin cancers. J Invest Dermatol. 2006;126:336–344. 36. Kogelnik AM, Lott MT, Brown MD, Navathe SB, Wallace DC. MITOMAP: a human mitochondrial genome database. Nucleic Acids Res. 1996;24:177–179. 37. Mita S, Rizzuto R, Moraes CT, Shanske S, Arnaudo E, Fabrizi GM, Koga Y, DiMauro S, Schon EA. Recombination via flanking direct repeats is a major cause of large-scale deletions of human mitochondrial DNA. Nucleic Acids Res. 1990;18:561–567. 38. Schon EA, Rizzuto R, Moraes CT, Nakase H, Zeviani M, DiMauro S. A direct repeat is a hotspot for large-scale deletion of human mitochondrial DNA. Science. 1989;244:346–349. 39. Hou JH, Wei YH. The unusual structures of the hot-regions flanking large-scale deletions in human mitochondrial DNA. Biochem J. 1996;318(Pt 3):1065–1070. 40. Fullerton SM, Bernardo Carvalho A, Clark AG. Local rates of recombination are positively correlated with GC content in the human genome. Mol Biol Evol. 2001;18:1139–42. 41. Scharffetter-Kochanek K, Wlaschek M, Brenneisen P, Schauen M, Blaudschun R, Wenk J. UV-induced reactive oxygen species in photocarcinogenesis and photoaging. Biol Chem. 1997;378: 1247–1257.
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31 DNA Damage and Repair in Skin Aging Daniel B. Yarosh
Introduction DNA has many roles in skin cell function, including directing metabolism, storing the information of heredity, and sensing cell danger. Damage to DNA is a major cause of the chronic conditions of aging and photoaging. The natural repair system offers significant protection, and new compounds offer the promise of augmenting DNA repair. This chapter focuses to a large extent on Ultraviolet (UV) damage to DNA because solar UV is by far the greatest danger to DNA. Sun exposure is a major public health concern, and has been directly linked to most of the (more than one million) new types of skin cancer that arise in the USA each year [1]. From 1990 to 1994, premalignant and malignant skin cancers accounted for 19% of visits to the dermatologist [2]. DNA damage caused by solar UV has been directly linked to these skin cancers, as the cancers contain suppressor genes mutations that are characteristic of UV in their inactivated tumor [3].
Sources of DNA Damage DNA damage comes from two sources: the intrinsic metabolism of the cell and the environmental insult. Intrinsic metabolism. During aerobic energy generation, about 2% of all the oxygen burned ends up as reactive oxygen species (ROS). DNA is damaged by ROS most frequently by the oxidation of the guanine base to form 8-oxo-guanine (8oGua), which is often misread by the DNA replication machinery, causing a mutation. This is particularly serious for mitochondria, whose DNA is closest to the source of the short-lived ROS. Environmental insult. By far, the most serious damage to skin DNA is from the sun. The cyclobutane pyrimidine dimer (CPD) is formed ten times more frequently than 8oGua, and is caused by direct absorption of UV photons without any ROS intermediate. A second type of base fusion, a 6–4 photoproduct, is similarly formed about one sixth as frequently as CPD. CPDs cause a
characteristic type of DNA mutation produced by no other carcinogen, and these signature mutations are frequently found in key cancer genes in squamous and basal cell carcinomas. This is the smoking gun that connects CPDs to skin cancer. Solar UV also causes the formation of ROS, which results in ROS and then 8oGua. In mitochondria, repeated doses of UVA result in the accumulation of a characteristic deletion mutation in mitochondrial DNA. The frequency of this characteristic mutation in human skin increases with sun exposure, suggesting that it is an internal dosimeter for cumulative sun exposure. Not all DNA damage is from sun exposure. Alkylation is a third type of DNA damage in which an alkyl group is added to DNA. The most prevalent additions are at the 7 position of guanine (N7-meGua) and to the phosphates of the DNA backbone, but a much less common form of damage, alkylation of the 6 position of guanine (O6-meGua) is the most mutagenic and hence the most dangerous. This type of damage is usually caused by side reactions of DNA with normal metabolites such as nitrites, and by toxins like some of the chemicals in cigarette smoke. The premature signs of aging in the skin in smokers may be associated with the alkylation damage from alkylating agents in cigarette smoke transported through the circulation to the skin. Additionally, exposure to other chemicals via exposure to industrial pollution and drugs with DNA alkylating potential may have some role in what has been considered normal skin aging.
Sun Damage to DNA Wavelengths of Sunlight that Damage DNA DNA readily absorbs photons in the UV portion of the solar spectrum. Although the shorter UVC wavelengths (200–280 nm) do not actually reach the earth’s surface due to their absorption by the ozone layer, the longer wavelength UVB (280–315 nm) is still relatively efficient in causing direct damage to the DNA bases and largely penetrates only into the epidermis [4]. The even longer
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_31, # Springer-Verlag Berlin Heidelberg 2010
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wavelength UVA (315–400 nm) penetrates into the dermis; however, since these photons carry less energy, they are relatively less efficient in producing direct damage to DNA than UVB and create proportionately more reactive oxygen species in the skin cells that indirectly damage DNA [5]. Recent evidence suggests that very high doses of visible light can produce indirect DNA damage through the formation of reactive oxygen species [6].
Photoproducts Solar UV directly causes an instantaneous photochemical reaction in DNA that links together adjacent pyrimidine bases (cytosine or thymine) [7]. This cyclobutane pyrimidine dimer (CPD) is the most common form of DNA damage, and is formed by all UV wavelengths, including UVA, UVB, and UVC [8]. After a sunburn dose, on the order of 100,000 CPDs are formed in the DNA of every sun-exposed cell. In a much less common reaction, solar UV can directly link together these bases by a single twisted bond, resulting in a 6–4 photoproduct (<6–4 > PP) [8]. Solar UV can also cause DNA damage by an indirect method, through the formation of reactive oxygen species that attack DNA, particularly the guanine base. This oxidation reaction most often results in 8-oxo-guanosine (8oGua), but even after UVA exposure CPDs are much more common than 8oGua [9]. Oxidation of DNA can
also result in single-stranded breaks, but under physiological conditions these are very difficult to detect. When single-stranded breaks are found after UV irradiation, they are almost all caused by DNA repair enzymes cutting the DNA in an intermediate step in repair.
Mechanisms of DNA Repair DNA is the rare biomolecule that is not discarded when it is damaged, but rather is repaired. Human cells have developed two fundamental repair strategies to restore DNA to its native sequence and conformation (> Table 31.1).
Nucleotide Excision Repair More than 20 different proteins participate in this multistep process, and many of these proteins also participate in RNA transcription and/or DNA synthesis. In a typical day a cell may have to repair 10,000 damaged bases and after sun exposure each cell of the skin may have to remove 100,000 lesions! This process consumes cellular stores of nicotinamide adenine dinucleotide (NAD), which are used to tag sites of single-stranded breaks and other damages. The depletion of NAD can endanger cell energy reserves, so niacin or niacinamide, members of the vitamin B family and precursors of NAD, are necessary to replenish the NAD reservoir.
. Table 31.1 DNA repair activities. The source of DNA damage and the major form of the damage is shown. The type of DNA repair, the key enzymes in the repair and the relative time for repair is also shown. The means for prevention of the lesions are also shown, including the use of sunscreens, and the natural melanin and antioxidants in skin Source
DNA damage
DNA repair
Key enzymes
Time
Protection
Solar UV
CPD
NER, global and transcription coupled
XPA-XPG
Slow
Sunscreen/Melanin
Solar UV
<6–4>
NER
XPA-XPG
Slow
Sunscreen/Melanin
Solar UV
CPD
Photoreactivators (non-mammation)
Photolyase
Fast
Sunscreen/Melanin
Reactive oxygen species
Single strand breaks
BER
DNA ligase
Fast
Sunscreen/Anti-oxidant
Reactive oxygen species
8-oxo Guanine
BER
OGG1
Fast
Sunscreen/Anti-oxidant
Reactive oxygen species
Double strand breaks
ATM
Slow
Sunscreen/Anti-oxidant
CPD Cyclobutane Pyrimidine Dimers; NER Nucleotide Excision Repair; XPA-XPG Xeroderma Pigmetosum complementation groups A through G; BER Base Excision Repair; OGG1 8-oxo-Guanine Glycosylase; ATM Ataxia Telangiectasia Mutant
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Major damage to DNA, such as CPDs or < 6–4 > PPs, interferes with its coding ability and must be repaired in order for the nucleotide sequence to function. Each of these is removed in a patch of about 30 DNA nucleotides by a process termed nucleotide excision repair (NER) [10]. A dozen or more proteins may cooperate to complete NER. One subset of these proteins recognizes CPDs throughout the genome because they distort the regular turns of the DNA helix, and initiate global genomic repair (GGR). However, an additional set of proteins is especially responsive to RNA transcription forks, which are stalled at sites of CPDs in the coding sequence, and are able to more quickly mobilize the NER machinery to these regions of DNA vital to cell function to initiate transcription coupled repair (TCR). Once these recognition proteins bind to the site of DNA damage, they recruit additional enzymes that unwind the DNA, make a single-stranded break on either side of the CPD, and release the 30-nucleotide piece of DNA. The single-strand gap is then filled in by DNA polymerases using the opposite strand of the DNA as a template. Each cell has several varieties of DNA polymerases and most of them copy DNA very accurately. However, a few types are much more error prone and when they are called into service, they introduce mutations by incorporating incorrect bases into the patch [11]. NER of CPDs is not a very efficient process. After UV exposure that produces a sunburn in human skin, it takes about 24h to remove 50% of the damage. NER repair of < 6–4 > PPs is much quicker: about 50% are removed in 30 min. This is due to the fact that < 6–4 > PPs are less frequent, and so they greatly distort DNA that they are easier for the NER proteins to locate and excise.
Base Excision Repair Damage to single bases such as 8oGua is much less distorting to DNA, and is repaired by a second pathway termed base excision repair (BER) [10]. Here a DNA repair enzyme termed oxo-guanine glycosylase-1 (OGG1) specifically recognizes 8oGua and releases it from the DNA backbone, leaving an abasic site. A second enzyme recognizes this baseless site and makes a single-stranded break. A few bases on either side of the break are removed and the short patch is again resynthesized using the opposite strand as a template. This is a speedy process, and half of the 8oGua introduced by solar UV are repaired in about 2h [12]. In human cells, CPDs are not repaired by BER because there is no glycosylase to recognize them. However, the
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bacteriophage enzyme T4 endonuclease V recognizes CPDs and clips one side of the dimer from the DNA, initiating BER. Amazingly, when delivered into human cells this enzyme functions quite well to initiate repair of CPDs by BER [13].
Photoreactivation An additional pathway of DNA repair is used by plants, fish, reptiles, and amphibians, but it is not present in humans or other mammals. This repair is accomplished by the enzyme photolyase that directly reverses CPD by capturing longwavelength UV and visible light, and using the energy to split the bonds that bind together the pyrimidine bases in a CPD [14]. This restores the DNA to normal without producing a single-strand break or removing any DNA. Once again, while human cells have no photolyase enzymes, when these enzymes are introduced into human cells they function quite well in repairing CPDs [15].
Diseases of DNA Repair Much has been learned by studying rare genetic diseases with defects in DNA repair and other diseases in which skin cancer rates are elevated. This has not only clarified the function of many of the DNA repair proteins, but has also revealed that many DNA repair proteins have multiple functions in the cell.
Xeroderma Pigmentosum, Trichothiodystrophy, Cockayne Syndrome Xeroderma pigmentosum (XP) is characterized by mildto-extreme photosensitivity, often with areas of hypopigmentation and hyperpigmentation, an increased risk of skin cancer and a shortened life expectancy [16]. There are seven complementation groups of XP (A–G), corresponding to defects in one of seven genes that code for proteins involved in NER, and a variant group with a defect in repair synthesis. Stringent photoprotection from an early age can greatly reduce actinic damage, but does not prevent neurological defects that are a hallmark of some of the complementation groups. This may be because some of these genes are also involved in non-DNA repair gene transcription. Trichothiodystrophy (TTD) patients have a defect in the same gene as XP-D patients, but at different locations within the gene, so they manifest photosensitivity, stunted growth, and brittle hair, but not an increase in
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skin cancer [16]. This highlights that subtle differences in a DNA repair protein can produce drastic differences in human development and morphology. Patients with Cockayne syndrome (CS) have mutations in one of two genes that code for proteins controlling TCR, and they also have growth and developmental abnormalities, but surprisingly little increased risk of skin cancer [16].
Solid Organ Transplant Patients Organ transplant patients have an elevated rate of skin cancer on sun-exposed skin during the period in which they are on immunosuppressive therapy [17]. There is no doubt that suppression of the immune system plays a significant role in allowing nascent skin cancers to grow out. However, there is increasing evidence that these drugs also impair DNA repair in the skin [18]. The two most widely used drugs, CsA and tacrolimus, target the phosphatase calcineurin. Calcineurin dephosphorylation of the nuclear transcription factor NFAT allows NFAT to localize in the nucleus, where it is a key activator of transcription of several immunoregulatory genes. Immobilization of calcineurin sequesters NFAT in the cytoplasm and shuts down transcription of these genes. Other transcription factors, such as TFHII, are vital to the preferential repair of DNA by targeting the repair machinery to sites of stalled transcription complexes. NFAT may also participate in recovery from transcription blocks.
DNA Repair Gene Polymorphisms The genes implicated in these genetic diseases code for proteins that participate not only in DNA repair, but also in other routine developmental programs and cell functions. The general population carries many forms of these genes with other, less serious, mutations, and these forms are called genetic polymorphisms. While some of these polymorphisms are innocuous, groundbreaking research shows that some gene forms increase the risk of cancer, including skin cancer [19]. One such DNA repair gene polymorphism is in the OGG1 gene coding for the glycosylase that releases 8oGua from DNA. The OGG1 polymorphism S326C has been associated with an increased risk of several types of cancer [20]. However, three separate in vitro biochemical studies of the activity of the protein produced by the variant gene failed to identify any deficit in activity or reduced DNA repair of oxidatively damaged DNA [21–23]. The S326C
variant polymorphism in the OGG1 gene is linked to increased risk of cancers such as prostate cancer, but the protein produced by the variant gene does not have any obvious biochemical defects. The variant polymorphic genotype however, is the most sensitive to cell killing by cytotoxic agents, and the heterozygous genotype was most resistant [24]. Delivery of exogenous OGG1 enzyme to cells increased repair of 8-oxo-guanine in the homozygous variants [25]. Thus, subtle changes in DNA repair genes may alter their activity in cells and increase susceptibility to endogenous and exogenous damage.
Prevention of DNA Damage Melanin The first line of defense against DNA damage is the pigment deposited by melanocytes at the surface of the skin. Melanocytes are pigment-producing cells that are found in the basal layer of the epidermis and disperse melanosomes, containing melanin, among the surrounding keratinocytes. These melanosomes encapsulate two main classes of pigments found in human skin: eumelanin, which is brown or black, and pheomelanin, which is reddishbrown. The relative amounts of these two pigments, and the size and density of the melanosomes largely determine the differences in skin color among humans. The constitutive pigment that is associated with racial groups, is deposited by melanocytes above the nuclei of keratinocytes, thereby shielding them from UV. Skin color has an enormous effect on the risk of skin cancer because this constitutive melanin absorbs and reflects a broad spectrum of UVR. Thus, UV exposure to dark skin produces less DNA damage than in light skin. The induced pigmentation in tanned skin, however, is significantly dispersed as pigment granules, rather than capping nuclei. The result is that tanned skin is much less protective against DNA damage than the equivalent in constitutive color.
Sunscreens Sunscreens are an additional front-line defense against DNA damage by reflecting or absorbing UV at the skin surface. The absorbed energy is released from the sunscreen molecules mostly as fluorescence or heat. Sunscreens are either inorganic, physical sunscreens that largely reflect light, or chemical sunscreens that mostly absorb light. Some sunscreens are less photostable than others
DNA Damage and Repair in Skin Aging
and lose their absorption capacity during UV exposure. Some of the energy absorbed by sunscreen molecules can cause the release of ROS, and this is true for both physical and chemical sunscreens. Recent advances in sunscreen development have been designed to reduce or eliminate these possibilities. To date there is no evidence that ROS released by sunscreens in skin causes significant levels of DNA damage. Of far greater concern is that sunscreens are usually not used properly or in the right amounts, and despite their application, significant DNA damage still results [26]. The most frequently used physical UV filters are the inorganic micropigments, zinc oxide, or titanium dioxide in the range of 10–100 nm in diameter. These micropigments are capable of reflecting a broad spectrum of UV rays in the UVA and UVB regions. Major disadvantages of micropigments are that they also reflect visible light, creating the so called ‘‘ghost’’ effect on skin, and they are difficult to formulate, often resulting in disagreeable preparations in which the micropigments have a strong tendency to agglomerate, which greatly decrease their efficacy. Chemical UV filters have the capacity to absorb shortwavelength UV photons and to transform them into heat by emitting long-wavelength photons (infrared radiation), which are much less likely to damage DNA. Most chemical filters absorb in a relatively small wavelength range. In general, chemical filters may be divided into molecules, which absorb primarily in the UVB region (290–310 nm) and those, which primarily absorb in the UVA region (320–400 nm). Only a few chemical filters absorb both UVB and UVA photons. Although these are relatively easier to formulate into cosmetically elegant textures, combinations of chemical filters are required in order to meet regulatory standards for sunprotection.
Antioxidants A third protection against the formation of DNA damage is antioxidants. They absorb ROS and thereby prevent oxidative DNA damage, primarily 8oGua. Because CPD are not formed by an ROS intermediate, antioxidants cannot prevent them. The natural skin antioxidant system is composed of lipophilic antioxidants such as vitamin E and CoQ10 and hydrophilic antioxidants such as vitamin C, glutathione, and the enzymes catalase and superoxide dismutase. An exciting new finding is that the powerful antioxidant ergothioneine and its receptor (OCNT-1) are found in the suprabasal layer of the epidermis, as
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well as in the dermis. This implicates ergothioneine as a new natural component of the human skin antioxidant system. Antioxidants cooperate to regenerate each other after reacting with ROS. For example, oxidation of vitamin C leads to its fast degradation, but vitamin E can generate oxidized vitamin C. In the same way, vitamin C can regenerate ergothioneine. Complete antioxidant protection requires many types of antioxidants, since ROS can be in the form of singlet oxygen, superoxides, or peroxides, as well as others. They can also be sequestered in the water or lipid compartment of cells. Therefore, the examination of the antioxidant protection system of skin requires consideration of all the antioxidants as a network.
Cellular Effects of DNA Damage A complex system regulates the cell’s progression through division to insure that only undamaged ones replicate, in order to avoid genetic instability and cancer [27, 28]. As cells approach commitment to DNA synthesis (S phase), proteins encoded by checkpoint genes delay entry if DNA damage is present. DNA proteins kinases, such as ATM (Ataxia-Telangiectasia Mutated) and ATR (AtaxiaTelangiectasia Mutated and Rad3 Related), then initiate signaling cascades resulting in DNA damage responses that include activation of the p53 protein. This tumor suppressor plays a central role in whether a cell repairs the damage [29] or is diverted into programmed cell death (apoptosis), cell cycle arrest, or senescence [27]. Mitochondrial DNA is damaged largely as a result of oxidative damage secondary to the production of excess ROS by UV or normal metabolism. Sufficient levels of this damage cause release of mitochondrial factors, such as cytochrome C, which binds to the apoptotic proteaseactivating factor 1 (Apaf-1), resulting in the formation of the apoptosome. This critical event leads to the activation of caspase-9 and the initiation of the mitochondrial apoptotic pathway through caspase-3 activation [30]. Apoptosis is a critical event preventing damaged cells from progressing to malignancy. One new photoprotection strategy is to selectively target DNA-damaged cells for apoptosis while leaving normal cells unaffected. Oral administration of caffeine or green tea (which often contains high levels of caffeine) in amounts equivalent to three to five cups of coffee per day to UVB-exposed mice increased levels of p53, slowed cell cycling, and increase apoptotic sunburn cells in the epidermis [31].
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Signal Transduction The dramatic events that follow DNA damage indicate that DNA is an important sensor of environmental insult and is able to trigger a variety of cell responses. The molecular mechanisms for this sensor-effector mechanism are being unraveled. The UV-induced cyclobutane pyrimidine dimers and pyrimidine (6–4) pyrimidone photoproducts cause distortions in the DNA helix and halt RNA polymerase II (RNA-PII) transcription of DNA. Protein kinases that activate their downstream targets through phosphorylation play an important role in signal transduction. A group of protein kinases that interact with DNA (ATR, Chk2, DNA-PK,) is implicated in the molecular cascade that detects and responds to several forms of DNA damage caused by genotoxic stress [32]. ATR (ATM-Rad3related kinase) is a primary DNA sensor and is essential for UV-induced phosphorylation of several G1/S checkpoint proteins. ATR was also shown to bind UVB-damaged DNA, with a resulting increase in its kinase activity, with many proteins as its target [33]. One such target is the RNA-PII itself, where phosphorylation represses further transcription initiation. This stalled RNA polymerase II leads to recruitment of the nucleotide excision repair complex. Another target of ATR is p53. Following phosphorylation, the half-life of the p53 protein is increased drastically, leading to a quick accumulation of p53 in stressed cells. Next, a conformational change forces p53 to take on an active role as a transcriptional regulator in these stressed cells. p53 is able to transactivate a plethora of genes with an active role in cell cycle arrest, global genomic DNA repair, apoptosis, and cytokine release.
Systemic Effects of DNA Damage Cytokines DNA in the skin acts like a sensor for UV damage on behalf of both exposed and unexposed cells in distal parts. DNA damage triggers the production and release of cytokines that act on the cell itself, as well as other cells with such cytokine receptors, to activate characteristic UV responses, such as wound healing and immune suppression [34]. Keratinocytes are the main source of these cytokines. Other epidermal cells, like Langerhans cells (LHC), and melanocytes, together with infiltrating leukocytes are also active contributors to changed cytokine profile after UV exposure. Keratinocytes are able to secrete a
wide variety of proinflammatory cytokines upon UV exposure, including interleukins IL-1a, IL-1b, IL-3, IL-6, IL-8, granulocytes colony-stimulating factor (G-CSF), macrophage-CSF (GM-CSF), interferon gamma (INF-g), platelet-derived growth factor (PDGF), transforming growth factor alpha (TGF-a), TGF-b, and tumor necrosis factor a (TNF-a) [35–37]. Cytokines such as IL-1 and TNF-a then induce a cascade of other cytokines that can activate collagendegrading enzymes, suppress the immune system, dilate blood vessels, and attract inflammatory T cells [38]. In this way, cells with DNA photodamage, even if they are destined to die, have profound effects on cells in the skin and elsewhere that may not have been exposed to UV. IL-12 plays a curious role in photoprotection. It is an immunostimulatory cytokine that is released by keratinocytes at late times after UV in order to counteract the suppressive effects of IL-10 [39]. Recently, it has also been reported to stimulate the repair of CPDs in the DNA of keratinocytes in a manner yet to be understood [40].
Immune Suppression UV-induced immune suppression is an essential event for skin cancer formation [41]. It is important to note that this is not a generalized immune suppression, but a reduced ability to respond to antigens presented just after exposure. There may be a genetic susceptibility to UV-induced suppression, because skin cancer patients are more easily UV suppressed than cancer-free controls [42]. At lower UV doses, the primary target is the Langerhans cells, which flee the epidermis and those with DNA damage have impaired antigen-presenting ability [43]. Higher doses produce systemic immune suppression, mediated by the generation of suppressor T cells, in which nonexposed skin becomes hampered in responding to antigens [41]. In several experimental models, including humans, reducing DNA damage decreases the degree of immune suppression [44].
Wound Healing and Photoaging UV-induced DNA damage also triggers a wound healing response in the skin, as it tries to eliminate damaged cells and stimulate cell division to replace them. UVR directly to fibroblasts, as well as signals from damaged keratinocytes, cause the release of metalloproteinase (MMP-1), which selectively degrade large collagen cables [45]. Soluble factors released by keratinocytes, including IL-1, IL-6, and TNF-a, are principle actors in this paracrine effect [46].
DNA Damage and Repair in Skin Aging
DNA damage is directly related to the release of soluble mediators since enhanced repair of keratinocyte DNA reduced the release of the mediators and lowered the release of MMP-1 by unirradiated fibroblasts [47]. As part of this response, MMP-2 and MMP-9, which are responsible for digesting small collagen fragments, are downregulated by UVR. This results in the accumulation of collagen fragments, which severs the anchorage of fibroblasts, inhibits their ability to produce new collagen, and degrades the dermal elastic fiber network [48]. This is followed by hyperproliferation among keratinocytes, and together these responses are designed to fill in sites of skin wounds. Repeated rounds of this type of imperfect wound healing produces many of the microscopic hallmarks of photoaged skin, including a corresponding decrease in the biophysical properties of the skin [49], reflected in a loss of both skin strength and elasticity, flattening of the rete ridges, and the appearance of wrinkles and skin folds. Additionally, there are degradative vascular changes in the dermis resulting in telangiectasia and decreases in the capillary network and skin blood flow [50]. These small changes accumulate after repeated rounds of DNA damage to form what is readily recognized as aged skin. These connections, from DNA damage to stalled transcription complexes, resulting in kinase cascades activating metalloproteinases, which degrade skin collagen, explain why photoaging is a product of unrepaired DNA lesions.
Mutations and Skin Cancer Mutations Most of the solar UV-induced DNA damage distorts the double helix. In attempting to replicate past CPD lesions the cell often makes the same mistake of misincorporating two consecutive bases, resulting in mutations characteristic of UV damage [51]. In many cases these mutations have no effect on the cell, but if they occur at critical locations in tumor suppressor genes, they abrogate apoptosis and initiate the process of carcinogenesis. These UV ‘‘signature’’ mutations are often found in mutated p53 genes, a key tumor suppressor gene, in human squamous cell and basal cell carcinomas [51]. This is the key link between UV exposure and skin cancer, and directly implicates CPDs in carcinogenesis. These p53 signature mutations are also frequently found in precancerous actinic keratosis, suggesting that these mutations are an early step in the process of forming squamous cell carcinomas,
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and that later steps, such as additional gene mutations and immune suppression, determine if a cell goes on to malignancy. The situation is less clear in melanoma. There appear to be many different tumor suppressor genes that can be mutated in melanoma, and the frequency of signature mutations is not as common as in squamous cell carcinoma [52]. Mitochondria generate energy for the cell, and they contain DNA that encodes many of the crucial proteins in the energy production machinery. This DNA is also subjected to mutations, and mitochondria develop a peculiar type of mutation called the common deletion, in which a particular 477 base pair section of the DNA is deleted. The frequency of the common deletion in the mitochondria of human skin cells does not correlate with chronological age, but rather with sun exposure and photoaging [53]. This implies that solar UV is responsible for the formation of the common deletion, and its contribution to the signs of photoaging is an active area of research.
Prevention of Skin Cancer with DNA Repair Enzymes The inevitable consequence of the accumulation of DNA damage over a lifetime is an increased incidence of mutations and an elevated risk of skin cancer. The primary strategy for reducing this risk is the attenuation of the UV dose striking the skin, by sun avoidance, pigmentation, and sunscreens. Antioxidants have become a part of the defense by scavenging ROS before they can oxidize DNA. The next step in intervention is the enhanced repair of DNA damage before it can be fixed as a mutation and increase the probability of malignant transformation. Over the past 40 years the field of DNA repair has identified many enzymes that recognize and initiate removal of DNA damage, either by nucleotide excision repair, base excision repair, or direct reversal. The use of some of these enzyme activities for photoprotection became practical with the development of liposomes specifically engineered for delivery into skin [54]. The small protein T4 endonuclease V from bacteriophage recognizes the major form of DNA damage produced by UVB, which is the cyclobutane pyrimidine dimer (CPD). Liposomal delivery of T4 endonuclease V to UV-exposed human skin increased repair from 10% of CPD to 18% over 6 h, but dramatically reduced or eliminated the release of cytokines such as IL-10 and TNF-a [55]. In a randomized clinical study of the effects of daily use of this liposomal
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T4 endonuclease V in XP patients, the rate of premalignant actinic keratosis and basal cell carcinoma was reduced by 68% and 30%, respectively, compared to the placebo control [56].
Conclusion During a lifetime, skin is exposed to chemical challenges generated by its own metabolism as well as the environment, particularly solar UV. There are a variety of defenses in the human body, such as skin color, sunscreens, and antioxidants, to counteract these. Inevitably, however, cells sustain damage. DNA serves the cell not only as the master controller of cell function, and the storehouse of heredity information, but also as a sensor of damage, and consequently a sentinel for danger to the cell and the organism. It is able to convert the distortion caused by altered nucleic acid bases into signals that arrest and redirect its own cell machinery. It also converts that distortion into notification of adjoining cells, whether damaged or not, that significant lesions have occurred. The purpose of these signals is to evoke repair and healing responses. DNA is a unique macromolecule in carrying with it the toolkit for its own repair. DNA repair is focused at the site of actively transcribed DNA by a complex of enzymes, some of which are specifically adapted to recognize modified DNA, and some borrowed from the transcription machinery itself. The repair may have the task of repairing hundreds of thousands of lesions daily, and while it is an efficient process it is not perfect. The resulting mutations in the DNA sequence are the necessary components for the development of skin cancer. Skin aging, therefore, can be viewed as the accumulation of imperfections from repeated rounds of DNA damage and repair, as well as rounds of wounding and healing. Skin cancer is just one manifestation of these cycles. Viewed in this way, it is likely that properly conceived efforts to alleviate skin aging will also have the benefit of reducing rates of skin cancer. Since over long periods of time, people are more motivated by improving their physical appearance than lowering their perceived risk of disease, the most successful anticancer efforts will arrive as treatments for skin aging.
Cross-references > Fibulin-5
Deposition in Human Skin: Decrease with Aging and UVB Exposure and Increase in Solar Elastosis
References 1. Housman T, et al. Skin cancer is among the most costly of all cancers to treat for the Medicare population. J Am Acad Dermatol. 2003;48:425–429. 2. Salasche S. Epidemiology of actinic keratosis and squamous cell carcinoma. J Am Acad Dermatol. 2000;42:S4–S7. 3. Brash D. Sunlight and the onset of skin cancer. Trends Genet. 1997;13:410–414. 4. Yarr M, et al. Photoaging: mechanism, prevention and therapy. Br J Dermatol. 2007;157:874–887. 5. Cadet J, et al. Ultraviolet radiation – mediated damage to cellular DNA. Mutat Res. 2005;571:3–7. 6. Mahmoud BH, et al. Effects of visible light on the skin. Photochem Photobiol. 2008;84:450–462. 7. Schreier W, et al. Thymine dimerization in DNA is an ultrafast photoreaction. Science. 2007;315:625–629. 8. Yoon J-H, et al. The DNA damage spectrum produced by simulated sunlight. J Mol Biol. 2000;299:681–693. 9. Courdavault S, et al. Larger yield of cyclobutane dimers than 8-oxo7, 8-dihydroguanine in the DNA of UVA-irradiated human skin cells. Mutat Res. 2004;556:135–142. 10. Sancar A, et al. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Ann Rev Biochem. 2004;73: 39–85. 11. Christmann M, et al. Mechanisms of human DNA repair: an update. Toxicology. 2003;193:3–34. 12. Yarosh D, et al. After sun reversal of DNA damage: enhancing skin repair. Mutat Res. 2005;571:57–64. 13. Tanaka K, et al. Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus). Proc Natl Acad Sci USA. 1975;72:4071–4075. 14. Carell T, et al. The mechanism of action of DNA photolyases. Curr Opin Chem Biol. 2001;5:491–498. 15. Stege H, et al. Enzyme plus light therapy to repair DNA damage in ultraviolet-B-irradiated human skin. Proc Natl Acad Sci USA. 2000;97:1790–1795. 16. Cleaver J. Cancer in xeroderma pigmentosum and related disorders of DNA repair. Nat Rev. 2005;5:564–573. 17. Randle H. The historical link between solid-organ transplantation, immunosuppression, and skin cancer. Dermatol Surg. 2004;30: 595–597. 18. Yarosh D, et al. Calcineurin inhibitors decrease DNA repair and apoptosis in human keratinocytes following ultraviolet B irradiation. J Invest Dermatol. 2005;125:1020–1025. 19. Au W, Navasumrit, et al. Use of biomarkers to characterize functions of polymorphic DNA repair genotypes. Int J Hyg Environ Health. 2004;207:301–304. 20. Goode E, Ulrich C, Potter J. Polymorphisms in DNA repair genes and associations with cancer risk. Cancer Epid Biom Prev. 2002;11: 1513–1530. 21. Kohno T, et al. Genetic polymorphisms and alternative splicing of the hOOG1 gene, that is involved in the repair of 8-hydroxyguanine in damaged DNA. Oncogene. 1988;16:3219–3225. 22. Dherin C, et al. Excision of oxidatively damaged DNA bases by the human a-hOGG1 protein and the polymorphic a-hOGG1 (Ser326Cys) protein which is frequently found in human populations. Nucl Acids Res. 1999;27:4001–4007.
DNA Damage and Repair in Skin Aging 23. Janssen K, et al. DNA repair activity of 8-oxoguanine DNA glycosylase I (OGG1) in human lymphocytes is not dependent on genetic polymorphism Ser326/Cys326. Mutat Res. 2001;486:207–216. 24. Yarosh D, et al. DNA repair gene polymorphisms affect cytotoxicity in the National Cancer Institute Human Tumor Cell Line Screening Panel. Biomarkers. 2005;10:188–202. 25. Yarosh D, et al. After sun reversal of DNA damage: enhanced skin repair. Mutat Res. 2005;571:57–64. 26. Mahroos R, et al. Effect of sunscreen application on UV-induced thymine imers. Arch Dermatol. 2002;138:1480–1485. 27. Funk JO. Cell cycle checkpoint genes and cancer. Encyclopedia Life Sci. 2005;1–5. 28. Harper JW, et al. The DNA damage response: ten years after. Mol Cell. 2007;28:739–745. 29. Sancar A, et al. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem. 2004;73: 39–85. 30. Guzman E, et al. Mad dogs, Englishmen and apoptosis: the role of cell death in UV-induced skin cancer. Apoptosis. 2003;8:315–325. 31. Lu Y-P, et al. Effect of caffeine on the ATR/Chk1 pathway in the epidermis of UVB-irradiated mice. Cancer Res. 2008;68:2523–2529. 32. Zhou BB, et al. The DNA damage response: putting checkpoints in perspective. Nature. 2000;408:433–439. 33. Unsal-Kacmaz K, et al. Preferential binding of ATR protein to UVdamaged DNA. Proc Natl Acad Sci USA. 2002;99:6673–6678. 34. Kondo S. The roles of keratinocyte-derived cytokines in the epidermis and their possible responses to UVA-irradiation. J Invest Dermatol Symp Proc. 1999;4:177–183. 35. Ansel J, et al. Cytokine modulation of keratinocyte cytokines. J Invest Dermatol. 1990;94:101S–107S. 36. Luger TA, et al. Evidence for an epidermal cytokine network. J Invest Dermatol. 1990;95:100S–104S. 37. Enk AH, et al. Early molecular events in the induction phase of contact sensitivity. Proc Natl Acad Sci USA. 1992;89:1398–1402. 38. Heck DE, et al. Solar ultraviolet radiation as a trigger of cell signal transduction. Toxycol Appl Pharmacol. 2004;195:288–297. 39. Barr R, et al. Suppressed alloantigen presentation, increased TNF-a, IL-1, IL-1RA, IL-10, and modulation of TNF-R in UV-irradiated human skin. J Invest Dermatol. 1999;112:692–698. 40. Schwarz A, et al. Interleukin-12 suppresses ultraviolet radiationinduced apoptosis by inducing DNA repair. Nature Cell Biol. 2002;4:26–31.
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41. Kripke M. Immunologic unresponsiveness induced by UV radiation. Immunol Rev. 1984;80:87–102. 42. Streilein J. Immunogenetic factors in skin cancer. N Engl J Med. 1991;325:884–887. 43. Vink A, et al. The inhibition of antigen-presenting activity of dendritic cells resulting from UV irradiation of murine skin is restored by in vitro photorepair of cyclobutane pyrimidine dimers. Proc Natl Acad Sci USA. 1997;94:5255–5260. 44. Kuchel J, et al. Cyclobutane pyrimidine dimer formation is a molecular trigger for solar-simulated ultraviolet radiation-induced suppression of memory immunity in humans. Photochem Photobiol Sci. 2005;4:577–582. 45. Brennan M, et al. Matrix metalloproteinase-1 is the major collagenolytic enzyme responsible for collagen damage in UV-irradiated human skin. Photochem Photobiol. 2003;78:43–48. 46. Wlaschek M, et al. UVA-induced autocrine stimulation of fibroblastderived collagenase/MMP-1 by interrelated loops of interleukin-1 and interleukin-6. Photochem Photobiol. 1994;59:550–556. 47. Dong K, et al. UV-Induced DNA damage initiates release of MMP-1 in human skin. Exp Dermatol. 2008;17:1037–1044. 48. Fisher GJ, et al. Looking older. Fibroblast collapse and therapeutic implications. Arch Dermatol. 2008;666–672. 49. Leveque J-C, et al. Aging Skin: Properties and Functional Changes. Aulnoy-sous Bois: Informa Health Care, 1993. 50. Ryan T. The ageing of the blood supply and the lymphatic drainage of the skin. Micron. 2004;35:161–171. 51. Brash D. Sunlight and the onset of skin cancer. Trends Genet. 1997;13:410–414. 52. High W, et al. Genetic mutations involved in melanoma: a summary of our current understanding. Adv Dermatol. 2007;23:61–79. 53. Berneburg M, et al. Induction of the photoaging-associated mitochondrial common deletion in vivo in normal human skin. J Invest Dermatol. 2004;122:1277–1283. 54. Yarosh D, et al. Localization of liposomes containing a DNA repair enzyme in murine skin. J Invest Dermatol. 1994;103:461–468. 55. Wolf P, et al. Topical treatment with liposomes containing T4 endonuclease V protects human skin in vivo from ultraviolet-induced upregulation of interleukin-10 and tumor necrosis factor-a, J. Invest Dermatol. 2000;114:149–156. 56. Yarosh D, et al. Effect of topically applied T4 endonuclease V in liposomes on skin cancer in xeroderma pigmentosum: a randomized study. Lancet. 2001;357:926–929
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Endogenous and Exogenous Factors in Skin Aging
41 Effect of Ozone on Cutaneous Tissues Giuseppe Valacchi
Introduction Living organisms are continuously exposed to environmental pollutants. Depending on their state, pollutants can be taken up by ingestion, inhalation, or contact with the skin. Because the skin is an interface between the body and the environment, it is chronically exposed to several forms of stress such as ultraviolet (UV) radiation and other environmental oxidants such as cigarette smoke and ozone (O3). UVB and, to a lesser degree, UVA induce various skin pathological conditions, including erythema, edema, hyperplasia, ‘‘sunburn cell’’ formation, photoaging, and photocarcinogenesis. There is abundant information that reactive oxygen species (ROS) such as hydroxyl radicals are involved in UV-induced skin damage, both by direct effects of UV and by subsequent phagocyte infiltration and activation. Oxidative environmental pollutants, such as cigarette smoke, O3, and oxides of nitrogen that have been studied in the respiratory tract [1], also represent a potential oxidant stress to the skin. In order of importance, the skin is the second most frequent route by which chemicals can enter into the body. The skin is the major target of liquid and gaseous pollutants and the pollutant that reacts most specifically with the cutaneous tissues beside UV radiation, hydrocarbon, and organic compounds in O3. Ozone represents one of the major oxidants in photochemical smog, levels being highest in heavily polluted areas where exposure to UV is also high. In the last decade, many studies have shown the toxic effect of O3 on the skin [2, 3].
a stable structure but exists in several mesomeric states in dynamic equilibrium.
In the liquid and solid states, O3 is highly explosive, and among oxidant agents, it is the third strongest (O3, E = +2.076 V) after fluorine (+3.0353 V) and persulfate (+2.866 V).
Ozone: The Good and the Bad O3 is naturally present in the atmosphere surrounding the Earth. In the upper part of the atmosphere, the stratosphere, 20–30 km from the earth’ surface, the O3 layer can reach the concentration of 10 ppm. The O3 occurring in the stratosphere, where the majority of atmospheric ozone is found, forms a ‘‘filtering layer’’ that acts as a barrier to the dangerous radiation from the sun.
Physicochemical Properties of Ozone The word ozone derives from the Greek word deo, which means ‘‘to give off a smell.’’ It is an unstable gas of a soft sky-blue color, with a pungent, acrid smell already perceptible at a concentration of 0.01 ppb. The molecule is composed of three oxygen atoms (O3) and has a molecular weight of 48 kDa. It has a cyclical structure assessed by the spectrum absorption in the infrared region, with a distance of 1.26 A˚ among oxygen atoms. O3 does not have
In contrast, O3 present within the lower troposphere (10 miles from the ground level) is hazardous and dangerous to the terrestrial health. It is an ubiquitous
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_41, # Springer-Verlag Berlin Heidelberg 2010
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pollutant of the urban environment, and it is not emitted directly by any man-made source in significant quantities. Ozone arises from chemical reactions in the atmosphere through the action of sunlight on oxygen molecules. The photochemistry involved in the generation of O3 usually involves several reactions such as photo activation, photodecomposition, and free radical chain reaction [4]. The most common molecules that lead to O3 formation at the ground level are nitric oxides (NOx). NO2 can be photolyzed by solar UV resulting in NO and the atomic oxygen that can react with molecular oxygen leading to the formation of O3. Ozone can also be destroyed by nitric oxide. NO can react with O3 to form NO2 and O2. Under these steady state conditions, the concentration of O3 cannot increase until most of NO has been converted to NO2 by additional reactions occurring within the complex. This accumulation occurs as the rate of NO2 photolysis is much faster than that of O3. Other species in photochemical smog also undergo photodecomposition to yield free radicals that may participate either directly or indirectly in the conversion of NO to NO2. Hydroxyl and hydroperoxyl radicals are the examples of compounds that can react with nitrogen radicals with the destruction of O3 by NO.
The average tropospheric amount of ozone ought to be far less than 40 ppb, which is much lower than that present in the stratosphere. Yet in large metropolises such as Mexico City, and also in European cities such as Rome, Milan, and Paris, O3 can reach toxic concentrations (0.8 ppm) especially during the summer. Anthropogenic emissions, mainly of NOx and also methane (CH4), carbon monoxide (CO), and sulfuric compounds, have caused a progressive increase of O3 concentration up to 1,000 ppb or more [5]. At the street level, O3 has become the main toxicant not only for the respiratory tract, but also for the cutaneous tissues.
Ozone in Life Reports of hazardous effects induced by smog reach as far back as the thirteenth century when, during the reign of Richard III (1377–1399), human diseases were attributed
to severe air pollution. Trends in tropospheric O3 are poorly documented. The O3 level in the northern hemisphere increased significantly during the periods of industrialization. In the late nineteenth century, O3 was measured near Paris (Montsouris) to follow a seasonal cycle and to be in the range of 10–20 ppb. Such values are considerably lower than present-day with background concentration values of 40–50 ppb observed over the continents in the Northern hemisphere. The presence of O3 in air was well recognized in Los Angeles during the early 1940s, based on its damaging effects on rubber products (http://www.who.int/en/). In the 1950s, London and other major cities in UK suffered a series of smog episodes that caused the death of 4,000 people in a week [6]. During pollution episodes, O3 mixing ratios higher than 80 ppb can be observed locally in the industrialized regions of North America, Europe, and Asia. It is likely that, on an average, the O3 abundance has increased by a factor of 2 or more since the preindustrial era (http:// www.who.int/en/), and it is, therefore, conceivable that oxidizing power of the atmosphere has changed during the same period. It has been estimated by Zeng and Pyle that the level of tropospheric O3 will be increased fivefold at the end of this century because of the increase of cars and industrial fumes, leading to dangerous consequences to the terrestrial life [7]. The average environmental O3 levels, that vary considerably for many reasons need to be known, in order to understand the effects of a daily 8-h O3 exposure (April–October). The US Clean Air Act has set an O3 level of 0.06 ppm (120 mg/m3) as an 8-h mean concentration to protect the health of workers (US Environmental Protection Agency, 2005). Evaluation of recent studies [8, 9] allows establishing an average environmental O3 concentration of 90 10 ppb. However, O3 concentration in urban air can exceed 800 ppb in high-pollution conditions [10], reducing not only pulmonary functions and enhancing the risk of cardiovascular death, but also affecting skin physiology.
Ozone as an Oxidant It is generally understood that, although O3 is not a radical species per se, the toxic effects of O3 are mediated through free radical reactions and they are achieved either directly by the oxidation of biomolecules to give classical radical species (hydroxyl radical) or by driving the radicaldependent production of cytotoxic, nonradical species (aldehydes) [11].
Effect of Ozone on Cutaneous Tissues
Furthermore, the formation of the oxidation products characteristic of damage from free radicals has been shown to be prevented by the addition of the antioxidant vitamins E and C, though the mechanism is not fully understood. The target specificity of O3 toward specific compounds together with its physicochemical properties of fairly low aqueous solubility and diffusibility must be taken into account when a target tissue like the skin is exposed to O3. As it was hypothesized [12], O3 does not penetrate the cells, but oxidizes available antioxidants and reacts instantaneously with surfactant’s polyunsaturated fatty acids (PUFA) present at the air–cellular interface to form reactive oxygen species (ROS), such as hydrogen peroxide and a mixture of heterogenous LOPs including lipoperoxyl radicals, hydroperoxides, malonyldialdeyde, isoprostanes, the ozonide radical, O3 [13], and alkenals, particularly 4-hydroxy-2,3-trans-nonenal (HNE) [14]. As cholesterol is a component of the upper layer of the skin and because its double bond is readily attacked by O3, it can give rise to biologically active oxysterols [15]. A major ozonation product of cholesterol, 3b-hydroxy-5-oxo-5,6-secocholestan6-al, induces apoptosis in H9C2 cardiomyoblasts of which 3b-hydroxy-5-oxo-5,6-secocholestan-6-al has been implicated in pulmonary toxicity, Alzheimer’s disease, and atherosclerosis. Cell membranes and their lipids are relevant potential targets of environmental stressors such as O3. Using a spin trapping technique, the formation of radicals in the SC upon exposure to O3 was detected. The spin adduct could arise from an alkoxyl radical formed during lipid peroxidation. Furthermore, lipid radicals (L●) are generated in epidermal homogenates that have been exposed to environmental stressors. The organic free radical L● reacts with O2, forming peroxyl radical LOO● and hydrolipoperoxides (LOOH). Transition metals, and in particular iron, play a key role in the reactions of LOOH and in the subsequent generation of alkoxyl radicals (RO● can amplify the lipid peroxidation process). Moreover, the toxicity is certainly augmented by the presence of NO2, CO, SO2, and particles (PM10). On this basis, it appears clear how the O3-generated ROS and LOPs at the tissue level, after being only partly quenched by the antioxidants, will act as cell signals able to activate transcription factors (nuclear factor-kappa B (NF-kB), NO synthase and some protein kinases, thus enhancing the synthesis and release of proinflammatory cytokines (TNFa, IL-1, IL-8, IFNg, and TGFb) and the possible formation of nitrating species. With a possible increasing inflow into the cutaneous tissues of neutrophils and activated macrophages, a vicious circle will start,
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perpetuating the production of an excess of ROS including also hypoclorous acid [16], LOPs, isoprostanes, tachykinins, cytokines, and proteases, which will self-maintain the inflammation after O3 exposure.
The Stratum Corneum as the First Target of Environmental Stressors Within the skin, the SC has been identified as the main target of oxidative damage. As the outer skin barrier, the SC has important functions, limiting transepidermal water loss and posing a mechanical barrier to penetration by exogenous chemicals and pathogens. It comprises a unique two-compartment system of structural, nonnucleated cells (corneocytes) embedded in a lipid-enriched intercellular matrix, forming stacks of bilayers that are rich in ceramides, cholesterol, and free fatty acids. The effects of O3 on cutaneous tissues have been evaluated using a murine model and in a few studies using even human subjects [17, 18]. While no effect of O3 on endogenous antioxidants was observed in fullthickness skin (dermis, epidermis, and SC), it could be demonstrated that a single high dose of O3 (10 ppm for 2 h) significantly depleted topically applied vitamin E. When the skin was separated into upper epidermis, lower epidermis, papillary dermis, and dermis, O3 induced a significant depletion of tocopherols and ascorbate followed by an increase in the lipid peroxidation measured as malondialdehyde (MDA) content. O3 is known to react readily with biomolecules and does not penetrate through the cells; therefore, it was hypothesized that O3 mainly reacts within the SC [19]. This hypothesis was supported by further experiments, where hairless mice were exposed to varying levels of O3 for 2 h. Depletion of SC lipophilic (tocopherols) as well as hydrophilic (ascorbate, urate, GSH) antioxidants was detected upon O3 exposure and it was accompanied by a rise in lipid peroxidation as an indicator of increased oxidative stress. Furthermore, a recent study has shown the increase of 4-hydroxylnonenal (4-HNE) content in murine SC using both Western blot and immunohistochemical analysis. Finally, the increase of protein oxidation was also shown in in vivo studies [19, 20]. It is well known that oxygen radicals and other activated oxygen species generated as by-products of cellular metabolism or from environmental sources like O3 cause modification of the amino acids of proteins and therefore modifying their functions. Besides the modification of amino acid side chains, oxidation reactions can also alter the protein cross-linking with peptides. Protein
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carbonyls may be formed by oxidative cleavage of proteins or by direct oxidation of lysine, arginine, proline, and threonine residues. In addition, carbonyls groups may be introduced into proteins by reaction with aldehydes (4-HNE) produced during lipid peroxidation generated as a consequence of O3 reactivity with the cutaneous tissues. This explains the use of carbonyls formation as a marker of oxidative stress. Protein oxidation has been also associated with skin aging and other skin pathologies. Thiele et al. were able to detect protein carbonyls formation in the upper layer of the stratum corneum (SC) and in whole skin homogenates exposed to environmental insults. The main protein oxidized was keratin 10 and it showed an increasing level of oxidation from the lower SC to the upper level. This protein oxidation gradient was inversely correlated with the gradient of the antioxidant vitamin E. There are studies that have shown that protein oxidation can be quenched by antioxidants such as tocopherol and thiols. Of note is the fact that the keratin in SC contained dramatically more carbonyl groups than the keratin present in keratinocytes, indicating that the baseline levels of keratin oxidation are considerably higher in the SC as compared to the epidermal layers. Furthermore, the oxygen partial pressure, a ratelimiting factor for the formation of reactive oxygen intermediates in the skin, decreases gradually from the outer to the inner SC layers. All these may have important implications in the desquamation process of the SC because of the role that the proteins in corneodesmosomes play in cell cohesion. It should also be taken into account that while protein degradation increases proteolytic susceptibility up to a protein-specific degree, further damage actually causes a decrease in proteolytic susceptibility and leads to cross-linking and aggregation [19, 20].
Skin Antioxidant Defenses To protect itself against oxidative stress, the skin is equipped with an elaborate system of antioxidant substances and enzymes that includes a network of redox active antioxidants. Antioxidant enzymes such as glutathione reductases and peroxidases, superoxide dismutases, and catalase interact with the low-molecular-weight antioxidant substances such as vitamin E isoforms, vitamin C, glutathione (GSH), and ubiquinol [21]. The presence of tocopherol, ascorbate, urate, and glutathione has been demonstrated in the SC [21]. Interestingly, the distribution of antioxidants in the SC is not uniform, but follows a gradient with higher
concentrations in deeper layers and decreasing concentrations toward the skin surface. This may be explained by the fact that SC layers move up in time as a part of the physiological turnover, and are replaced by newly differentiated keratinocytes. Therefore, the superficial layer has been exposed longer to chronic oxidative stress than a deeper layer. Compared with the SC, the surface lipids contain high levels of tocopherol due to the secretion of vitamin E by sebaceous glands [22]. Eventually, the uppermost layer of the SC desquamates and the remaining antioxidants and reaction products will be eliminated from the body. In general, the outermost part of the skin, the epidermis, contains higher concentrations of antioxidants than the dermis. In the lipophilic phase, tocopherol is the most prominent antioxidant, while vitamin C and GSH have the highest abundance in the cytosol.
Ozone-Induced Cellular Responses in Cutaneous Tissues Inflammatory Responses Ozone, like many other environmental challenges, is able to activate redox-sensitive transcriptional factors such as nuclear factor kappa B (NF-kB). This transcriptional factor acts as an activator for a multitude of proinflammatory genes (IL-8, TNFa, and TGFb) and adhesion molecules (ICAM and VCAM). It has been assessed that O3 is able to activate NF-kB using both in vitro and in vivo systems. Thiele et al., using a immortalized human keratinocytes (HaCaT cells) was able to show that O3 induced the activation of NF-kB by electrophoretic mobility shift assay (EMSA). O3 induced a dosedependent activation of the transcription factor. This effect was likely to be mediated by ROS because it was inhibited by the incubation of the cells with lipid soluble antioxidants (tocopherol). Finally, using a murine model, an increase of proinflammatory marker cyclooxygenase-2 (COX-2) was detected confirming the role that O3 can play in skin inflammation [17].
Induction of Heat Shock Protein As mentioned above, O3 exposure was shown to induce antioxidant depletion as well as lipid and protein
Effect of Ozone on Cutaneous Tissues
oxidation in the SC. Recent studies have investigated the effects of O3 in the deeper functional layers of the skin. To evaluate the effect on cutaneous tissues of O3 exposure, hairless mice were exposed for 6 days to 0.8 ppm of O3 for 6 h/day and the skin responses were analyzed using the whole skin homogenates. Under these experimental conditions an increase in the protein level of heat shock protein (HSP)32, also known as hemoxygenase-1 (HO-1), confirming that HSPs are sensitive markers of O3-induced stress in cutaneous tissues. The author’s group was the first to document the upregulation of HSPs 27, 32, and 70 in homogenized murine skin upon O3 exposure. HSP27 showed the earliest (2 h) and highest (20-fold) response to O3 compared with the delayed induction (12 h) of HSP70 and HO-1. Increased expression of HSP27 has been demonstrated following heating of both keratinocyte cell lines and organ-cultured human skin. HSP27 is expressed predominantly in the suprabasal epidermis in human skin, whereas HSP70 predominates in the dermis compared with the epidermis. These differences in location between HSP27 and HSP70 might explain the different time course of induction of these stress proteins upon O3 exposure. Interestingly, O3 induction of HO-1 showed a delayed time course compared with that for HSP27 and 70, in line with a previous study, which showed a peak of HO-1 induction at 18–24 h in rat lungs after O3 treatment [17]. It is therefore possible that bioactive compounds generated by the products of O3 exposure may be responsible for the induction of HO-1 as was also shown after UV radiation. As HSPs are involved in cell proliferation, apoptosis, and inflammatory response, O3-mediated HSPs induction can affect normal skin physiology. Thus, HSPs might provide an adaptive cellular response to O3; enhancing the expression of HSPs might turn out to be a new way to deal with the immediate and long-term consequences of O3 exposure. A prerequisite for the utilization of this concept is the development of nontoxic HSP inducers and their evaluation for clinical efficacy and safety.
Ozone and MMPs Among the multiple systems altered in the skin by environmental pollutants, MMPs are among the major targets. Indeed, O3 exposure is able to affect their synthesis and/or activity with logical consequences on tissue remodeling and wound healing. Within the MMP family, MMP-2 and MMP-9 are the only members able to degrade type-IV
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collagen of the basal membranes [23]. MMP-2 is involved in pathological processes such as photoaging and precancerous/cancerous skin lesions after UV exposure; moreover, MMP-2 is capable of cleaving other substrates, in addition to type-IV collagen, including other MMPs and therefore can (indirectly) control extracellular matrix degradation and remodeling. MMP-9, like MMP-2, plays a role in human skin aging and tumor development as well as in other cutaneous lesions such as psoriasis and dermatitis [24]. In a recent study, it was demonstrated that the environmental pollutant O3 was able to affect specific types of MMP activity in whole skin homogenates from hairless mice. Specifically, the exposure to 0.25 ppm of O3 for 4 days (6 h/day) clearly induced MMP-2 activity in cutaneous tissues. In this case, the generation of ROS can be the cause of such activation, as it has been shown that MMPs can be activated by reactive oxygen species. It has been also demonstrated that O3 is able to induce NO production via the activation of iNOS in cutaneous tissues. NO, while playing regulatory roles in the skin at physiological levels, when produced in excess, may combine with superoxide to form peroxinitrite (derived from other sources) that can activated MMPs especially MMP-9. Thus, the increase of oxidative stress after O3 exposure, plus the interaction between oxygen and nitrogen active molecules might be the main mechanism that leads to the enhanced MMPs activities in skin tissues. It has been shown in a number of cases that photoaging and precancerous/cancerous lesions can result from an imbalance between MMPs and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) [24]. In fact the activities of MMPs are regulated by TIMPs, which can be produced by a multitude of cell types present in the cutaneous tissue. While MMP activity resulted to be altered by the O3, neither TIMP-1 nor TIMP-2 level expression was affected. The lack of changes in TIMP-1 and 2 levels, combined with the increased activity of MMPs suggest that O3 can cause a net increase in matrix degradation. Furthermore, there are other MMPs involved in skin diseases; for example, MMP-12, the human macrophage metalloelastase, accumulates in skin granuloma and in other inflammatory skin diseases such as dermatitis herpetiformis and pityriasis lichenoides. Moreover, MMP-7 or Matrilysin is very efficient in elastin degradation and increased elastolytic activity by both MMP-12 and MMP-7 has been reported upon oxidative stress exposure in hairless mice skin. Enhanced MMP-7 expression has also been detected in benign sweat gland tumors and aggressive basal and squamous cell carcinomas.
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Skin Age-Related Responses to O3 Exposure: MMPs It is well known that oxidative stress occurring after oxidant stressor such as O3 or UV radiation is implicated in the pathogenesis of skin-related diseases, and that the levels of antioxidant defenses decrease with aging. Consistently, it has been reported that aged mice are more susceptible to oxidative stress than young mice [25] and previous reports have suggested that oxidant pollutant exposure and age interact and potentiate each other [20]. Therefore, it can be concluded that among the multiple consequences of oxidative stress, an increase in the MMP/TIMP ratio also occurs. The question of whether a cause–effect relationship exists between oxidative stress and MMPs release, or they are two independent responses is not still clear and needs further studies to be better demonstrated. However, by integrating the results from recent works, the redox-associated signal transduction pathways that lead to MMPs induction can be easily reconstructed. Indeed, oxidative stress, through both receptor and non-receptor protein tyrosine kinases (PTK) activates several signaling proteins, such as ERK and PKB, which, in turn, mediate the transcriptional regulation of MMPs, via Ap-1, Ets, and NF-kB [26]. In closing, it is now documented that the interaction between aging and oxidative pollutant exposure can impair resistance of cutaneous tissues to environmental oxidative stress in elderly subjects.
Skin Age-Related Responses to O3 Exposure: Wound Healing Wound healing is a critical process in the skin and has been known to be affected by oxidative stress and also to decline with increasing age. Although the exact sequence of wound healing is not completely understood, cutaneous wound healing begins with wounding induced signaling factor-based transformation of stationary keratinocytes into cells capable of both replication and migration. Upon transformation, these cells express a host of molecules that promote the invasion of the injured epithelial matrix and re-epithelialization of the wound surface. Delayed wound healing in the elderly has been well described [27]. Among the elderly, the SC transit time was delayed 10 days compared to young adults. More recently, Hellemans and coworkers published that older skin, subjected to UVA-induced deactivation of catalase, requires a longer time to replenish
the antioxidant capacity than in younger skin, furthermore, it was shown that aged skin strongly differs from young skin in the ability to cope to oxidative environmental insults [20–28]. In the recent literature, it has been shown that hydrogen peroxide (H2O2) (molecules involved in the induction of oxidative stress) induced vascular endothelial growth factor (VEGF) expression in human keratinocytes and therefore can be able to stimulate wound healing [29]. As mentioned above, O3 exposure is also associated with activation of transcription factor NF-kB, which is important to regulate inflammatory responses and eventually entire wound healing. O3 exposure increased levels of tumor growth factor (TGFb) that is a critical factor in tissue remodeling [30]. The roles of the multiple, coordinated processes involved in the injured skin repair, as well as the signals that initiate and terminate skin responses remain ill defined. Furthermore, the age-related differences in the response of the skin wound healing to particular environmental insults are poorly documented. Given the documented role of oxidants in wound healing [29], the potential effects of O3 on cutaneous wound healing in combination with aging represent a poorly understood area. It has been suggested that O3 as an oxidant might also stimulate wound healing but aging with O3 would be detrimental due to increased oxidative stress and have biological as well as practical implications. In a recent study the detrimental effects of O3 on cutaneous wound healing in the aged animals was demonstrated. In fact when young hairless mice (8-week old) and aged mice (18-month old) with full-thickness excisional wounds were exposed to 0.5 ppm O3 for 6 h/day the rate of wound closure was significantly delayed in the aged group. It was also shown that O3 exposure induces protein and lipid oxidation assessed as changes in protein oxidation (carbonyls) and lipid peroxidation (4-hydroxynonenal, HNE adducts) in the old mice compared to the young mice during the later stage of cutaneous wound healing. The extent of wound closure in young and old animals with full-thickness excisional wounds exposed to a relevant concentration of O3, was monitored until day 9 (complete wound closure) [20]. These data suggest that O3 exposure has different effects depending on the age of the mice. In fact, it significantly delayed wound closure in old mice, while in young mice, it had no significant effect, although an accelerated trend during the first few days of the exposure was detected. This might be attributed to the antibacterial properties of O3, as it has been shown that application of hydropressive
Effect of Ozone on Cutaneous Tissues
ozonization provides fast cleansing of wound surface from pyonecrotic masses, promotes elimination of infection, and thus substantially reduces the period of treatment of the patients. Recently, clinical treatments using hyperbaric oxygen therapy demonstrated that increased oxygen tension at the wound site increases the formation of granulation tissue and enhanced accelerated wound closure and ameliorated impairs dermal wound healing; therefore, accelerated trend of wound closure shown in young population may be due to decreased bacterial infection and/or increased oxygen tension by O3 exposure in wound area [31]. One of the possible driving processes of the effect of O3 on wound healing can be, also in this case, the modulation of the transcription factor NF-kB. Although NF-kB is an immune regulator in inflammatory stage, it maybe critical to modulate later stage of healing process in injury. Consistent with this one, a recent study reported that human airway epithelium inflammatory response to inhaled O3 has been shown to be in part controlled by free radical-mediated NF-kB activation. Further, very recently, it has been reported that overexpression of superoxide dismutase not only prevents O3-related changes in bronchoalveolar lavage fluid protein, macrophage content, and 4-hydroxyalkenals, but also O3-dependent activation of NF-kB [32]. These researchers have also reported that O3-induced lung injury is mediated by NF-kB. These results clearly link O3 exposure to NF-kB activation and suggest that intracellular oxidants such as superoxide and related free radicals are important components of these responses. Interestingly, the dose– effect relationship between level of oxidative stress and NF-kB exhibits a biphasic profile: while moderate levels of oxidative stress activate NF-kB through an IkB kinaseindependent mechanism, extremely high levels of oxidative stress have been shown to inhibit NF-kB activation by blocking IkBa phosphorylation. Furthermore, the levels of oxidative stress were increased in aged rats and the content of activated forms, p50, and p65 subunits of NF-kB increased with age. One potential explanation for the differential effect of O3 in the older animals is that the level of oxidative stress generated by O3 exposure combined with aging causes levels of oxidative stress that inhibits IkBa phosphorylation, thereby resulting in a decline in NF-kB activation. The existence of a higher basal level of oxidative stress in old mice is proved by the higher levels of protein carbonyls and 4-HNE. These data fit with studies that have shown that old rats had higher lipid peroxides and superoxide dismutase activity tended to decrease. This finding is consistent with what mentioned previously that O3 exposure induced skin antioxidants
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depletion. In addition, Gilhar and coworkers have reported that human epidermis showed reduced proliferation and increased keratinocyte apoptosis with aging [33]. This could be interpreted as an additional evidence of increased load of oxidative stress burdens in the keratinocytes of old mice, as apoptosis has been linked to elevated levels of cellular oxidants. The evidences and considerations reported above are controversial because the levels of cutaneous oxidative stress in response to O3 treatment should be higher in aged skin and these levels may be further increased by O3 exposure so as to raise levels of skin oxidative stress in old mice to levels above those that maximally evoke NF-kB activation. This study suggests that although O3 exposure increased NF-kB activation in the young and old mice it may differently modulate wound healing process by aging. Furthermore, NF-kB also have important roles in later tissue remodeling stage as well as in initial inflammatory stage during cutaneous wound healing. This interpretation is also bolstered by the data on TGFb, a crucial modulator of tissue remodeling and is linked to both NF-kB status and levels of oxidative stress during entire wound healing process. The reduced TGFb levels in both air- and O3-exposed old mice, as well as the lower induction of TGFb by O3 exposure in the old animals suggest that the noted delays in wound closure might be related to defects in oxidative stress-dependent NF-kB status as well as levels of oxidative stress and TGFb signaling in aged mice during later stage of wound healing. In summary, given the role of oxidative stress in wound healing, an interaction between O3 and aging is of great interest to be explored in cutaneous wound healing process. The ability of O3 to alter wound healing indicates that environmental effects of pollutants need to be taken into account when damaged skin repair is explored in human subjects.
Ozone Potentiates UV-Induced Oxidative Stress in the Skin Although exposure of cutaneous tissues to either UV or O3 alone is known to deplete vitamin E and induce lipid peroxidation, it is of interest to evaluate the possible additive effects of sequential or simultaneous exposure of skin to these important environmental oxidants stresses. It should be taken into consideration that the skin is continuously exposed to several environmental pollutans each day and UVand O3 are among the most toxic and noxious of them. While UV radiation penetrates into the epidermis (UVB) or into the dermis (UVA), and is known to induce the release of tissue-degrading enzymes even at
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suberythemal levels, O3 oxidizes biological systems only at the surface. Therefore, since O3 and UV cooperatively damage SC components, it can be speculated that they exert an additive effects in the deeper layers of the epidermis. Products of O3-induced lipid oxidation penetrate the outer skin barrier and cause effects to constituents of the deeper epidermis. This can lead to the activation of NF-kB. On the other hand, NF-kB activation has also been implicated in the expression of collagenases by solar-simulated UV radiation and in cutaneous responses to wounding. UV radiation has been shown to compromise the skin barrier. O3 may enhance this phenomenon by perturbing SC lipid constituents in the SC, which are known to be critical determinants of the barrier function. Thus, O3 may cause a disturbance of the barrier function, increase the transepidermal water loss, and provokes epidermal repair responses, as can be also seen after barrier perturbation. Since O3 enhances UV-induced oxidation in the SC, it cannot be excluded that potentially O3 also enhances other UV effects such as photoaging [34]. In conclusion, the ‘‘additive’’ data demonstrate that O3 and UV radiation, two common sources of environmental oxidant stressors, exhibit additive effects in terms of oxidative damage to the skin barrier.
Health Implication Being lipids, the first target of O3, the consequent induction of lipid peroxidation in the upper layers of the skin can affect the physiology of cutaneous tissues. In fact, oxidation of the lipids present in the SC will change the skin barrier integrity and this has been shown to be a leading factor for several skin pathologies such as psoriasis, atopic dermatitis, and irritant dermatitis. The increase of peroxidation markers such as 4-HNE, MDA, and TBARS in the upper layers of the skin after O3 exposure is a consequence of the PUFA peroxidation like arachidonic acid and linolenate and this could consequently affect also the lower layers of the skin trigging all a cascade of noxious biological processes. The toxicity of O3 is the results from the effects of a cascade of products that are produced in the reactions of O3 with target molecules that lie close to the air–tissue interface. Ozone is too reactive to penetrate far into the tissue, only a small fraction of environmentally relevant doses of O3 is believed to pass through a bilayer membrane, and none pass through the cell. Therefore, the products that derive from the oxidation of the SC, which have longer lifetime and lower reactivity will transmit the effect of O3 beyond the air–tissue interface. Peroxidation products such as
4-HNE and alkenals are relatively stable and can damage or alter cells and tissues at more distant sites not directly exposed to O3.
Therapeutical Approaches Because the SC is the main target of O3 reactivity, therapeutic strategies should involve the more accessible skin layers via a topical antioxidant application. In a murine model, topical application of vitamin E reduced the peroxidation induced by O3 exposure, demonstrating that topical application could be a way to counteract ozone-induced skin toxicity. This indicates a key role for vitamin E both as an indicator and in the prevention of skin oxidative damage. In addition to physical or chemical measures for protection against environmental stressors consistent with the ‘‘free radical theory of aging,’’ the use of low-molecularweight antioxidants for preventing premature skin aging and skin disease seems appropriate. Vitamin E and other antioxidants can only be supplied to the skin to some extent via a diet rich in fruits and vegetables. Moreover, vitamin E supplementation and/or its topical administration will substantially enhance skin vitamin E concentrations. Since oxidant skin alteration occurs mainly in the SC and outer epidermal layers, this is relevant for a preventive and/or therapeutic approach.
Conclusion The results summarized in this chapter support the concept first advanced by Pryor et al. [4] that O3 exposures to noncellular constituents of surface epithelial cells are capable of generating potentially toxic peroxidation products. Extrapolation of this concept to cutaneous tissues would suggest that O3 reacts directly with SC lipids that contribute to cutaneous tissue protective barrier, generating products that are able to penetrate the SC and target keratinocytes. It is concluded that O3 not only affects antioxidant levels and oxidation markers in the SC, but also induces cellular responses in the deeper layers of the skin (> Fig. 41.1a, b). It is recognized that exposure of the skin to environmental stressors causes injury to the skin due to oxidants and free radicals, which leads to ‘‘oxidative stress,’’ also defined as imbalance between oxidants and antioxidants. Low-molecular-weight antioxidants are present in high concentrations especially in the epidermis. Oxidative stress can overwhelm the skin antioxidants and increase
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. Figure 41.1 (a) Summarizing scheme showing the effects of ozone exposure to the cell membrane of the Stratum Corneum and consequently generation of bioactive compunds such as aldehydes and H2O2, etc that can transmit the oxidative effects to the deeper layer of the skin. (b) Scheme showing the possible events as a consequence of SC oxidation by Ozone exposure
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the formation of oxidized cell components. Topical exposure to tropospheric O3 induces skin oxidative stress. Oxidative damage to the SC may result in a barrier perturbation, and in the production of lipid oxidation products that can act as ‘‘second messengers’’ in the deeper layers of the skin, which, in turn, elicits repair responses and/or the induction of defense enzymes such as HSPs. Oxidative injury to the outermost layers of the skin may initiate localized inflammatory responses, resulting in the recruitment of phagocytes and their cell-specific, tightly regulated NAD(P)H-oxidase systems for generating oxidants, thus amplifying oxidative stress and inducing activation of MMPs.
Cross-references > Global
Warming and its Dermatologic Impact on Aging Skin > Skin Photodamage Prevention: State of the Art and New Prospects
References 1. Kelly FJ, et al. Air pollution and the elderly: oxidant/antioxidant issues worth consideration. Eur Respir J Suppl 2003;40:70s–75s. 2. Valacchi G, et al. Induction of stress proteins and MMP-9 by 0.8 ppm of ozone in murine skin. Biochem Biophys Res Commun. 2003;305 (3):741–746. 3. Valacchi G, et al. The dual action of ozone on the skin. Br J Dermatol. 2005;153(6):1096–100. 4. Mudway IS, et al. Ozone and the lung: a sensitive issue. Mol Aspects Med. 2000;21(1–2):1–48. 5. Zimmermann PH. Tracing the sources of tropospheric ozone, Proceedings of the International Ozone Symposium, 21 and 22 October 1999. Basel (IOA – EA3G Ed), 1999, pp. 157–160. 6. Weber SU, et al. Ozone: an emerging oxidative stressor to skin. Curr Probl Dermatol. 2001;29:52–61. 7. Dentener F, et al. The global atmospheric environment for the next generation. Environ Sci Technol. 2006;11:3586–3594. 8. Mortimer KM, et al. The effect of ozone on inner-city children with asthma: identification of susceptible subgroups. Am J Respir Crit Care Med. 2000;162(5):1838–1845. 9. Tager IB, et al. Chronic exposure to ambient ozone and lung function in young adults. Epidemiology. 2005;16(6):751–759. 10. Mustafa MG. Biochemical basis of ozone toxicity. Free Radic Biol Med. 1990;9(3):245–265. 11. Pryor WA. Mechanisms of radical formation from reactions of ozone with target molecules in the lung. Free Radic Biol Med. 1994;17(5):451–465. 12. Pryor WA, et al. The cascade mechanism to explain ozone toxicity: the role of lipid ozonation products. Free Radic Biol Med. 1995;19(6):935–941.
13. Ballinger CA, et al. Antioxidant-mediated augmentation of ozoneinduced membrane oxidation. Free Radic Biol Med. 2005;38 (4):515–526. 14. Esterbauer H, et al. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med. 1991;11 (1):81–128. 15. Sathishkumar K, et al. A major ozonation product of cholesterol, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al, induces apoptosis in H9c2 cardiomyoblasts. FEBS Lett. 2005;597(28):6444–6450. 16. Spickett CM, et al. The reactions of hypochlorous acid, the reactive oxygen species produced by myeloperoxidase, with lipids. Acta Biochim Pol. 2000;47(4):889–899. 17. Valacchi G, et al. In vivo ozone exposure induces antioxidant/stressrelated responses in murine lung and skin. Free Radic Biol Med. 2004;36(5):673–681. 18. He QC, et al. Effects of environmentally realistic levels of ozone on stratum corneum function. Int J Cosmet Sci. 2006;28(5):349–357. 19. Thiele JJ, Schroeter C, Hsieh SN, et al. The antioxidant network of the stratum corneum. Curr Probl Dermatol. 2001;29:26–42. 20. Lim Y, et al. Modulation of cutaneous wound healing by ozone: differences between young and aged mice. Toxicol Lett. 2006;160(2):127–134. 21. Packer L, et al. Antioxidants and the response of skin to oxidative stress: vitamin E as a key indicator. Skin Pharmacol Appl Skin Physiol. 2002;15(5):282–290. 22. Thiele JJ, et al. Sebaceous gland secretion is a major physiologic route of vitamin E delivery to skin. J Invest Dermatol. 1999;113 (6):1006–1010. 23. Brenneisen P, et al. Ultraviolet-B irradiation and matrix metalloproteinases: from induction via signaling to initial events. Ann N Y Acad Sci. 2002;973:31–43. 24. Hofmann UB, et al. Matrix metalloproteinases in human melanoma. J Invest Dermatol. 2000;115(3):337–344. 25. Stadtman ER. Role of oxidant species in aging. Curr Med Chem. 2004;11(9):1105–1112. 26. Galis ZS. Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly. Circ Res. 2002;90 (3):251–262. 27. Crooks A. How does ageing affect the wound healing process? J Wound Care. 2005;14(5):222–223. 28. Fortino V, et al. Cutaneous MMPs are differently modulated by environmental stressors in old and young mice. Toxicol Lett. 2007;173(2):73–79. 29. Sen CK, et al. Oxidant-induced vascular endothelial growth factor expression in human keratinocytes and cutaneous wound healing. J Biol Chem. 2002;277(36):33284–33290. 30. Valacchi G. Studies on the biological effects of ozone: 11. Release of factors from human endothelial cells. Mediators Inflamm. 2000;9(6):271–276. 31. Gajendrareddy PK, et al. Hyperbaric oxygen therapy ameliorates stress-impaired dermal wound healing. Brain Behav Immun. 2005;19(3):217–222. 32. Fakhrzadeh L. Ozone-induced production of nitric oxide and TNFalpha and tissue injury are dependent on NF-kappaB p50. Am J Physiol Lung Cell Mol Physiol. 2004;287(2):L279–285. 33. Gilhar A, et al. Ageing of human epidermis: the role of apoptosis, Fas and telomerase. Br J Dermatol. 2004;150(1):56–63. 34. Valacchi G, et al. Ozone potentiates vitamin E depletion by ultraviolet radiation in the murine stratum corneum. FEBS Lett. 2000;466(1):165–168.
45 Environmental and Genetic Factors in Facial Aging in Twins David J. Rowe . Bahman Guyuron
Introduction The etiologic factors contributing to facial senescence have been investigated for decades if not centuries. In essence, the causes of facial aging can be divided into two broad categories: intrinsic and extrinsic [1]. Intrinsic aging is that which occurs as a response to the deterioration of tissues over time [2]. This process is ubiquitous throughout all organs and tissues, although the methods of ‘‘deterioration’’ may vary from system to system. Intrinsic changes of the face include those to the skin, subcutaneous tissue, dermal appendages, facial musculature, as well as the facial skeleton. The process of extrinsic facial aging is, theoretically, a distinct entity. Typically, extrinsic aging is induced by external factors, such as UV radiation, causing progressive damage at both the molecular and cellular levels. Unlike intrinsic aging, most extrinsic factors exert their effect at the skin level only. Discriminating between the levels of involvement of both aging processes, extrinsic and intrinsic, is problematic. As both processes continually occur, the ability for the body to resist change depends on the amount of cumulative damage of each individual process. Furthermore, there may be a dynamic interplay between extrinsic and intrinsic modalities that lead to variable amounts of aging depending on the levels of each and the ability of the body to repair or resist these. For example, a minor intrinsic defect, such as that in a DNA repair mechanism, may cause slightly accelerated aging from an intrinsic standpoint; however, it could cause an uncommonly aberrant response for extrinsic factors. A preponderance of information exists on the presence and types of extrinsic factors that may influence aging; however, the intrinsic causes are likely as important or more important on skin and facial aging. Multiple genetic disorders exist that corroborate the fact that premature aging can be genetic. Diseases with defects in DNA repair mechanisms, such as xeroderma pigmentosa, Werner Syndrome, and Cockayne Syndrome, all show accelerated aging [3]. Hutchinson-Gilford Progeria Syndrome is caused by a defect in Lamin A, a protein used in
the cell nuclear envelope, leading to abnormal morphology in the cell nucleus. This too leads to an accelerated aging phenotype. Theoretically, variable penetrance, or a less severe genetic aberration, may either increase or decrease the signs of aging, independent of external factors. Most data regarding the influence of external factors on aging have been conducted as epidemiological collections on specific patient populations. Epidemiological studies have intimated associations between environmental factors and aging; however, the influence of genetic differences cannot be controlled when dealing with a diverse genetic population. One research tool that may be implemented in the study of skin aging is the investigation of twin sets. Examination of monozygotic, as well as dizygotic twins, allows a unique opportunity to control for genetic differences. In the analysis of facial senescence, this gives the investigator the ability to control for most of the causes of intrinsic aging. Despite the relative benefit of genetic control, the statistical study of twins requires a relatively large patient population, as many twin sets have very similar lifestyles. The rest of the chapter attempts to analyze the findings of the several twin investigations in the current literature and identify possible risk factors for extrinsic aging.
Extrinsic Causes of Aging Smoking The association of skin aging with smoking was first scientifically proposed in 1856 when Solly postulated that facial appearance in smokers is markedly different than nonsmokers [4]. Since this time, epidemiological data have shown a possible correlation between smoking and facial aging, although the amount of associated aging due to smoking has been debated. Results have been equivocal, with several investigations showing little to no aging difference due to smoking status [5, 6] while others have seen a significant association [7–12]. Several analyses of twins have corroborated the influence of smoking on facial aging (> Fig. 45.1). Doshi et al.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_45, # Springer-Verlag Berlin Heidelberg 2010
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reported a significant difference in facial aging of a singular set of twins [13]. Each of the twins in this report had similar lifestyles, body habitus, and sun exposure; however, one of the twins had a greater than 50 pack-year history of smoking. The differences with regards to superficial and deep rhytides, lentigines, tissue laxity, and pigmentary changes were disparate. Antell and Taczanowski also provided anecdotal evidence on 34 sets of identical twins, showing a possible correlation to smoking differences [14]. Rexbye et al., in an investigation of 1826 Danish twin sets over the age of 70, demonstrated that smoking was a significant determinant of facial aging in men, yet less so in women [15]. Smoking 20 cigarettes per day per year for 20 years increased perceived age by one year. For women, this number was increased to 20 cigarettes per day for
. Figure 45.1 Twins (natural age 52) with difference in smoking history. Twin a, c had a 20 year greater smoking history than Twin b, d. Perceived age difference of the twins was 6.25 years
40 years (> Fig. 45.2). The authors attribute this discrepancy to the difference of smoking habits between the sexes in the Netherlands. Fewer women were smokers, and those who did smoke, actually smoked less than their male counterparts. Guyuron et al. conducted an investigation of 186 sets of identical twins at an annual international festival for twins [16]. Females from the ages of 18 to 76 were analyzed in this study. A comprehensive questionnaire was filled out and standardized high resolution photographs were taken of each twin set. Smoking was found to be significantly correlated with perceived facial age. In analysis of the data, approximately 10 years of smoking difference led to a 2 ½ years older appearance.
. Figure 45.2 Twins (natural age 57) with difference in smoking history. Twin b, d had a 40 year greater smoking history than Twin a, c. Twin a, c had 2 years of hormone replacement therapy. The perceived age difference was 8.25 years
Environmental and Genetic Factors in Facial Aging in Twins
The mechanism for smoking induced damage in the skin is unclear, although many theories exist. One postulate is that the increase of matrix metalloproteinases (MMPs) in the skin of smokers contributes to skin damage [17]. Matrix metalloproteinases are proteases responsible for degrading dermal collagen and other extracellular matrix material. This effect may be synergistic with the effects of UV radiation, as irradiation also induces MMPs. Other likely changes include the decrease of the skin microvasculature, leading to the increase of reactive oxygen species, and thus free radicals [13, 18]. These processes are also known to occur in the face of photodamage, therefore, there may be additive effects of sun and smoking on skin aging.
Sun Exposure Sun exposure is perhaps the most investigated cause of extrinsic aging. There is incontrovertible evidence that ultraviolet irradiation damages the skin and induces premature aging. The pathophysiology of photodamage is multifactorial, involving the upregulation of matrix metalloproteinases, reversible and irreversible damage to DNA, and creation of reactive oxygen species [18]. Details on these and other mechanisms are beyond the scope of this chapter and are discussed in subsequent chapters. Controversy does exist, however, on the importance of sun damage on perceived age. Chronological or intrinsic skin aging shares many of the features and possible mechanisms of photoaged skin. In both photoaging and chronological aging of the skin, elevated concentrations of degraded collagen are present. Furthermore, both mechanisms of aging have been theorized to occur as a result of oxidative damage [19]. Although the actual pathways involved in chronological aging may be distinct from those in photoaging, the two may share common central mediators. Many studies have been performed evaluating the importance of photoaging with respect to skin changes and alteration of perceived age [20]. While there is no question that UV irradiation induces age-related changes in the skin, the amount of change is a topic of considerable debate. When evaluating an elderly (> 60 years) population, Leung and Harvey found that sun exposure alone did not have a considerable effect on perceived age [2]. In their multivariate regression analysis, 30 years of sun exposure for 5 hours a day only produced 1.5 years of perceived age difference. Guinot et al., in designing a skin age score in a prospective analysis of Caucasian women from 18 to 80 years of age, found that visual signs of chronic
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photodamage did not contribute significantly to the age score [21]. These investigations do not refute the contribution of ultraviolet irradiation in skin aging but they do underscore the difficulty in attributing visible skin changes to a singular extrinsic or intrinsic cause. One method to investigate the importance of photoaging, and control for chronological (intrinsic) aging, is the analysis of identical twins. The analysis of twin sets has shown significant perceived differences in aging with respect to sun exposure (> Fig. 45.3). Guyuron et al. investigated hours of sun exposure as well as the participation in outdoor hobbies and the use of sunscreen [16]. The increase in sun exposure as well as the participation in outdoor activities both significantly increased perceived
. Figure 45.3 Twins (natural age 61) with significant difference in sun exposure. Twin b, d had approximately 10 h/week greater sun exposure than twin a, c. Twin a, c had a BMI 2.7 points higher than twin b, d. The perceived age difference was 11.25 years
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age. The use of sunscreen did significantly decrease perceived age; however, the level of SPF used was not assessed. Sun exposure was also correlated with increased perceived age in a study of elderly (70+) Danish twins [15]. Here, sun exposure was evaluated on the type of employment the individual performed during the longest period of working. Interestingly, sun exposure was statistically significant in men, however not in women. The authors attributed this effect to the likely high rate of sun exposure in working men. Only 8.3% of women in this study were exposed to sun during the working hours of the day.
. Figure 45.4 Twins (natural age 58) with differences in BMI. Twin a,c had a 14.7 point higher BMI than twin b, d. No other differences were discerned from the questionnaire. Perceived age difference was 5.25 years
Diet (BMI) BMI may have a not-so-indirect effect on perceived age, although this may not necessarily be due to changes at the skin level. For years, plastic surgeons have recognized that facial atrophy and soft tissue descent are harbingers for an increase in perceived facial age. Various surgical and injectable modalities have been introduced that restore facial volume and resuspend tissues to their previous anatomical position. Several studies have identified an inverse relationship between BMI and facial wrinkling. Guinot et al. prospectively analyzed a cohort of 361 white females in the process of developing a skin age score [21]. They found that body mass index did significantly affect their skin age score in an inverse relationship. Purba et al. evaluated skin wrinkling in elderly (> 70) patients from multiple countries [22]. As with the previous study, this investigation found that wrinkling was significantly negatively correlated with BMI. Analysis of twins’ data has further substantiated these findings. In Guyuron et al.’s study, the influence of body mass index was highly dependent on the age of the twin set [16]. In twins that were younger than 40 years of age, a four-point increase in BMI was associated with a perceived older appearance. A four-point increase in BMI in twins older than 40 years of age, however, was associated with a perceived younger appearance (> Fig. 45.4). The latter finding was supported by the Rexbye et al. investigation [15]. Here, an increase of BMI in both elderly men and women was associated with a younger appearance. A decrease of BMI of 2 in males and 7 in females was associated with a one year older perceived age.
Hormone Replacement Therapy (HRT) Estrogens have a profound effect on the skin, as evidenced by the cutaneous alterations in skin characteristics
following menopause. Although the mechanisms are poorly understood, a decrease in circulating estrogen is associated with decreased skin elasticity, decreased dermal thickness, as well as increased dryness [23]. Conversely, analyses of estrogen replacement therapy have suggested possible increases in the dermal thickness, concentration of dermal collagen, elasticity, and fewer fine wrinkles [24]. Epidemiological investigations have nonetheless been equivocal in their findings of hormone replacement therapy on perceived aging. With regard to twins’ analysis, hormone replacement therapy has been associated with a younger perceived age [16]. In addition, the effect of HRT on age increased as the age of the twin sets increased and as the difference of years of treatment increased (> Fig. 45.5).
Adverse Social Factors External social factors have been found to significantly alter biological aging. Several aspects that have been associated with aging include depression, divorce, socioeconomic status, and alcohol consumption [25, 26]. Additionally, there is usually an interaction or intermingling of these deleterious social factors. Osler et al. investigated the role of marital status, BMI, depression, and smoking in 1,175 sets of identical Danish twins [27]. The twins that were divorced, widowed, or never married had
Environmental and Genetic Factors in Facial Aging in Twins
. Figure 45.5 Twins (natural age 71) with difference in HRT. Twin b, d had 22 more years of HRT than twin a, c. Twin b, d had a 1.2 lower BMI. Perceived age difference was 7.25
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past, led to an increase in perceived age [16]. Rexbye et al. used a depression symptomatology score to analyze depression in their study [15]. They found that an increase in the depression score was significantly associated with an increase in perceived age. Specifically, an increase in depression symptomatology score from 17 to 49 was associated with a perceived age increase of 2.4 years for men and 3.9 years for women. The length and type of depression, however, has not been investigated with relation to facial aging. In both of these studies, only the presence of depression, either in the past or current was analyzed.
Alcohol Consumption A significant amount of data on the effects of alcohol on twins does not exist. Guyuron et al. investigated alcohol avoidance, but did not quantify alcohol consumption [16]. Despite this caveat, the twins who ‘‘avoided alcohol’’ had a younger perceived age. Rexbye et al. did not notice a significant effect of substantial alcohol consumption, however their number of positive responders was small enough to limit the power of this study [15].
Marital Status
higher depression scores and smoked more than their married counterparts. It is no surprise that perceived facial aging may also be adversely affected by these same factors. Limited epidemiological data exists on perceived facial aging and skin aging relating to factors such as depression, divorce, and alcohol consumption. However, several of the twins investigations previously discussed in this chapter have evaluated these social facets and will be discussed below.
Depression In the Guyuron et al. study, depression was indirectly investigated by measuring the utilization of antidepressants. Here, the use of antidepressnats, either current or
In the analysis of twins, women who were divorced were perceived to be approximately 1.7 years older than those who were either single or married [16]. Interestingly, widows appeared approximately two years younger than their non-widowed counterparts. Although Rexbye et al. also reported a 1.9-year difference in age between married and unmarried women, this was not statistically significant.
Conclusion The etiological delineation of skin and facial aging is complicated, given the large number of possible contributing factors. Epidemiological data has been helpful, however it does not allow for control of the intrinsic factors of aging that are inherently different in all individuals. The analysis of twins gives the investigator the ability to control for genetic causes. In the study of skin and perceived facial aging, one is able to investigate purely the environmental causes of skin and facial aging, extrinsic aging. From the data presented in the several twins investigations in the current literature, there appear to be several
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factors important to extrinsic skin aging and perceived facial age. Smoking and sun exposure, the two most epidemiologically studied factors, do appear to have a significant role in perceived age. Other factors such as hormone replacement therapy, BMI, depression, and the use of alcohol also have an influence on facial aging. Larger series of twins’ analyses are needed to further delineate the importance of each of these facets.
Cross-references > Effect
of Ozone on Cutaneous Tissues Warming and its Dermatologic Impact on Aging Skin > In Vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model > Skin Photodamage Prevention: State of the Art and New Prospects > Tobacco Smoke and Skin Aging > Global
References 1. Uitto J. Understanding premature skin aging. New Eng J Med. 1997;337:1419–1428. 2. Leung W, Harvey I. Is skin ageing in the elderly caused by sun exposure or smoking? Br J Dermatol. 2002;147(6):1187–1191. 3. Pesce K, Rothe M. The premature aging syndromes. Clin Dermatol. 1996;14:161–170. 4. Solly S. Clinical lectures on paralysis. Lancet. 1856;130(2):167–173. 5. O’Hare P, et al. Tobacco smoking contributes little to facial wrinkling. J Eur Acad Dermatol Venereol. 1999;12(2):133–139. 6. Allen H, Johnson B. Diamond S Smokers wrinkles? JAMA. 1973;225: 1067–1069. 7. Daniell H. A study in the epidemiology of ‘crows feet’. Ann Intern Med. 1971;75(6):873–880. 8. Model D. Smoker’s face: an underrated clinical sign? BMJ. 1985;291 (6511):1760–1763. 9. Ernster V, et al. Facial wrinkling in men and women, by smoking status. Am J Public Health. 1995;85:78–82.
10. Keadunce D, et al. Cigarette smoking: risk factor for premature facial wrinkling. Ann Intern Med. 1991;114(10):840–844. 11. Chung J, et al. Cutaneous photodamage in Koreans: influence of sex, sun exposure, smoking, and skin color. Arch Dermatol. 2001;137 (8):1043–1051. 12. Helfrich Y, et al. Effect of smoking on aging of photoprotected skin: evidence gatheres using a new photonumeric scale. Arch Dermatol. 2007;143(5):397–402. 13. Doshi D, Hanneman K, Cooper K. Smoking and skin aging in identical twins. Arch Dermatol. 2007;143(12):1543–1546. 14. Antell D, Taczanowski E. How environment and lifestyle choices influence the aging process. Ann Plast Surg. 1999;43:585–588. 15. Rexbye H, et al. Influence of environmental factors on facial ageing. Age Ageing. 2006;35(2):110–115. 16. Guyuron B, et al. Factors contributing to the facial aging of identical twins. Plast Reconstr Surg. 2009;123:1–11. 17. Lahmann C, et al. Matrix Metalloproteinase-1 and skin ageing in smokers. Lancet. 2001;357:935–936. 18. Fisher G, et al. Mechanisms of photoaging and chronological skin aging. Arch Dermatol. 2002;138(11):1462–1470. 19. Sohal R, Weindruch R. Oxidative stress, caloric restriction, and aging. Science. 1996;273:59–63. 20. Fisher G, et al. Pathophysiology of premature skin aging by ultraviolet light. New Engl J Med. 1997;337:1463–1465. 21. Guinot C, et al. Relative contribution of intrinsic vs extrinsic factors to skin aging as determined by a validated skin age score. Arch Dermatol. 2002;138(11):1454–1460. 22. Purba M, et al. Can skin wrinkling in a site that has received limited sun exposure be used as a marker of health status and biological age? Age Ageing. 2001;30:227–234. 23. Pierard G, et al. Effect of hormone replacement therapy for menopause on the mechanical properties of the skin. J Am Geriatr Soc. 1995;43(6):662–665. 24. Callens A, et al. Does hormonal skin aging exist? A study of the influence of different hormone therapy regimens on the skin of postmenopausal women using non-invasive measurement techniques. Dermatology. 1996;193(4):289–294. 25. Nilsson P, et al. Adverse social factors predict early ageing in middleaged men and women: the Ebeltoft Health Study, Denmark. Scand J Public Health. 2003;31(4):255–260. 26. Demakakos P, et al. Socioeconomic status and health: the role of subjective social status. Soc Sci Med. 2008;67(2):330–340. 27. Osler M, et al. Marital status and twins’ health and behavior: an analysis of middle-aged Danish twins. Psychosom Med. 2008;70 (4):482–487.
Rheology
27 Facial Skin Rheology Ge´rald E. Pie´rard . Fre´de´rique Henry . Pascale Quatresooz
Introduction The precise determination of the physical properties and functions of facial skin and its constituent parts remains an open question. In contrast, much more attention has been paid to the molecular biological characterization of skin components. In part, this may reflect the comparatively late development of bioengineering and biophysics and the intrinsic difficulty of obtaining relevant reproducible physical data from the skin. In addition, some ambient environmental conditions profoundly and specifically influence the physical attributes of the skin. As a result, the variations in physical parameters owing to body region, age, gender, and ethnicity greatly outweigh the variability of the corresponding molecular composition of the cutaneous structures. The term properties of the skin implies assessments similar to any other physical material, and this may give relatively little information about clinical or biological relevance. It is most applicable to in vitro testing. By contrast, clinicians and cosmetologists are primarily concerned with a more restricted range of functions rather than properties of the skin. It is of course true that functions are largely dependent on properties, but the conceptual and practical differences are important. Testing of a function must be performed in vivo under a fairly narrow range of ambient conditions. However, it does not follow that in vitro testing has no importance for clinicians. By contrast, some data are only obtained by using the in vitro approach, although this requires caution in interpretation.
Structure and Mechanical Functions of Skin Skin is a complex five-layered composite structure (stratum corneum, stratum Malpighi, papillary dermis, reticular dermis, and hypodermis), whose functions depend on the mutual interdependence of the constituent tissues. Overall, the mechanical properties and functions of skin are mainly governed by the dermal and hypodermal connective tissues, with a possible minimal contribution from
the stratum corneum [1, 2]. In addition, the skin structures differ largely according to the body site [1, 3]. Thus, regional variations have profound influences on the biomechanical characteristics. Moreover, the chronic and cumulative environmental threats including ultraviolet light and near infrared radiations [4] are dissimilar in distinct body regions, and according to age, phototype, and behavior with regard to sun exposure. The balance between these factors determines and distinguishes the intrinsic and extrinsic aging processes [5]. In these respects, facial skin is particularly susceptible to the diverse aspects of weathering and photoaging. Thus, aging of facial skin is not similar to that occurring on most other body sites. Such regional anatomical variation was not always fully acknowledged in the past. The mechanical properties and functions of skin are time dependent. They are also anisotropic as they differ according to the direction in which the load is applied. Both these characteristics of skin add complications in obtaining descriptors of its mechanical properties and functions. A further major problem in this field is the lack of standardization among investigators. Different research groups have used a variety of devices and measuring units, as well as different conditions of measurement. In addition, they have employed differing test modes for obtaining what was expected to be a similar information. It is clear that progress in the field of mechanical bioengineering will not be made until some attempt at uniformity is made, and acceptable, controlled, and standardized practices are developed.
Facial Skin Aging and Its Physical Attributes Facial skin is bound to deeper structures, but is allowed some mobility. Thus, it allows both movements and temporary compression and distension of a part. Flexibility and elasticity are important attributes of skin while firmness is also an essential component. These skin qualities are relevant to the visual and tactile features of facial skin [6]. Aging of facial skin takes place gradually over 2–4 decades. In its early stages, little clinical evidence is
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_27, # Springer-Verlag Berlin Heidelberg 2010
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present with the exception of the mosaic faint (subclinical) melanoderma [7]. The incipient clinical signs of facial aging usually begin to be recognized by individuals with the emergence of discrete furrows and wrinkles, together with a loss of firmness [8–11]. Additionally, various cutaneous signs and lesions develop with aging, and some of them are the result of more severe photoaging. In addition, changes in the deep cutaneous tissues distinct from sun-induced damages are responsible for deepening of facial creases and sagging [10–13]. As a rule, the results given by in vivo mechanical testing of facial skin may prove to be difficult to interpret because of the multiple and complex relationships between the various components of the skin, and the illdefined intrinsic properties of each tissue. In addition, there is commonly an unknown influence of previous mechanical solicitations at the test sites [1]. Both the mechanical functions and the surface contours of the skin reflect the structural organization of the tissues [10]. As a result, the combination of rheological and profilometric assessments provides a relevant and noninvasive characterization of the overall aging process of facial skin [1, 14, 15]. However, the diversity of bioengineering methods of evaluation combined with a wide variation in experimental designs has brought a number of uncertainties and discrepancies in the information.
Fibrous Structures Mainly Responsible for the Mechanical Properties On a mechanical viewpoint, the dermis has often been compared to rubber, and a series of the tests used were adaptations from those used in the rubber industry. In fact, the dermis does not exhibit similar properties and functions. The bulk of the cutaneous connective tissue consists of a network of collagen fibers, the organization of which determines the mechanical characteristics of the tissue and its resistance to deformation [1]. The elastic fibers present in smaller amounts serve to recoil the stretched collagen bundles to their relaxed position. This complex network of fibers is permeated by highly hydrated proteoglycans, glycoproteoglycans, and glycoproteins embedding the connective tissue cells. Fibroblasts and dermal dendrocytes are responsible for maintaining or remodeling the quantitative and structural steady state of the connective tissue fibers and the amorphous matrix as well. There are considerable differences in the relative proportions and organization of each of these components in different skin regions, as well as variations during aging and diseases [1].
With respect to the major mechanical functions of the connective tissues, and to structural features similar to all body regions, the dermis is conveniently divided into two major superposed layers. The adventitial dermis corresponds to a superficial zone of loose connective tissue adjacent to the epidermis and encasing its follicular adnexae as well. It corresponds to the papillary and the periadnexal dermis. The rest of the dermis is identified as the reticular dermis because of the netlike appearance of its fiber bundles. Still a third deeper layer corresponds to the connective septae partitioning lobules of adipocytes in the hypodermis. The differences and limits between the three layers are not always sharply identified. It is the relative concentration and arrangement of fibers rather than any absolute differences in composition that enable these regions to be distinguished. In mechanical terms, it is expected that the physical functions of the adventitial dermis somewhat resemble those of the hypodermal septae because they are conditioned by thin collagen fibers arranged in a rather similar loose open meshwork running perpendicular to the surface of the skin. The reticular dermis is more rigid because the collagen fiber bundles are coarser, tightly connected each another, and most often closely packed in planes parallel to the skin surface. It must be emphasized that this descriptive view of the connective tissue varies tremendously according to the body site. In its structural organization, the dermis of the face, scalp, back, forearm, palms, and soles differs greatly. It should be stressed that the rheological functions of facial skin are markedly influenced by the presence of a high density of terminal hairs in men and by abundant and large sebaceous glands. These structures likely put the surrounding fiber networks of the dermis under tension. In addition, facial muscles impose some anisotropic tensions to the skin, which are in turn responsible for some wrinkles such as facial frown lines and glabellar rhytids [9, 10, 16]. Moreover, age is important to consider as the skin presents marked differences during fetal life, childhood, climacteric period, and senescence [3, 14, 17]. Nonionizing radiations from the environment [4] superimpose their effects on those of the natural chronological aging [5, 18].
Physiological Interferences with Skin Mechanical Properties Skin withstands forces originating from the body and reacts to those imposed by the environment. These features govern the global skin mechanobiology. The perception of normal, loose, or tight skin depends on the
Facial Skin Rheology
ability of the connective tissue to resist and transmit the various forces. When assessing the in vivo skin mechanical functions, intrinsic tension forces are hardly measurable, and they should ideally be reduced to a minimum in order to prevent interference with testing [1]. This is tentatively achieved by muscle relaxation and a comfortable, controlled posture of the concerned body region. These controlled conditions do not abrogate the relaxed skin tension lines and the Langer’s lines [19–21]. Facial skin is quite unique as far as skin mechanical functions are concerned [14, 22–24]. Skin is anisotropic with regard to the variability of mechanical functions according to the direction of the forces applied [19, 25]. There is some complexity associated with sorting out some straightforward relevant information from most in vivo testings. In fact, measuring the overall mechanical characteristics of skin provides a rough estimate of the resultant of multiple features acting on various parts of the skin. The distinction between the specific dermal and epidermal properties is not accessible to accurate measurement. One of the major challenges resides in the interpretation of the combination of histological, instrumental, and biological variations that are found [1, 19]. In all circumstances, when a force is applied to the dermis, fibers are first reoriented in parallel to the force. At completion, some elongation is obtained for elastic fibers, while collagen fibers remain almost inextensible. In the physiological range of tension, the structural organization of collagen bundles, their orientations, the anchorage of bundles together, as well as their relation to elastic fibers and proteoglycans should be considered as predominant in determining the natural tension lines in the skin [20]. When skin is elongated, the fibers become aligned and slip over one another. With increasing forces the extensibility of the collagen fibers themselves is being tested. With sustained forces, there is a gradual change in the bonding of the collagen fibers or some other form of molecular realignment. During persistent compression, the interfiber matrix is squeezed out of the site. There is both an alignment of the fiber bundles along the lines of stress and a decrease in their convolution [20]. This first phase is largely due to straightening of the usually convoluted fibers but not to their lateral contraction.
Basic Viscoelastic Properties of Skin Basically, skin exhibits viscoelastic functions and properties. However, the literature may appear quite confusing with regard to the expression and interpretation of
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mechanical testing of the skin. This situation is due to the absence of a well-recognized nomenclature, of a uniform system of units, and of standardized techniques. However, the basic terms and units used in physics should be applied to biology, and these prove to be useful in clinical practice [1]. A series of experimental devices have been developed in research laboratories in order to measure the mechanical functions of the skin perpendicular or parallel to the skin surface. Mechanical functions of facial skin are conveniently assessed noninvasively by a series of methods. Skin may be pulled upward, pressed, twisted, extended parallel to its surface in one or several directions, and submitted to vibrations and to many other types of mechanical stimuli. Forces applied to the skin vary in direction, intensity, and time of application. These different approaches provide different information on the functions of the various connective tissue frameworks. In practice, the experimental approaches to determining the mechanical properties of skin are divided into six types: the tensile and torsional types, as well as those based on elevation, indentation, suction, and vibration. In general, the most relevant information is gained with testing facial skin at low stress. When a force is applied to the skin using a conventional testing device, the tension created is calculated in newtons (N) or in millibars (mbar). Stress corresponds to the ratio between the force (load) and the cross-sectional area of the skin in a plane at right angles to the direction of the force. It is expressed in newtons per square meters (N/m2). Strain is the ratio between elongation and the original length of the tissue submitted to the force. It is dimensionless, since measured as mm per mm. Usually, the crude information received from an experiment is the relationship of force (or stress) to deformation (or strain) over time. However, the maximum deformation for a given force is not gained immediately as some elongation still takes place under stable traction after certain periods of time. In addition, the deformation is not completely reversed within a short period of time in the absence of compressive force. These features explain the complexity of stress–relaxation curves. Facial skin is a viscoelastic material characterized by a nonlinear stress–strain properties with hysteresis (HY) [1, 26]. This means that the stress–strain curve obtained during loading is not identical to the curve obtained during unloading. Furthermore, the deformation of skin as a function of time shows an immediate incomplete elastic deformation and a creeping viscoelastic deformation followed by an immediate elastic recovery and a creeping recovery with a residual deformation. In any
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instance, the accuracy, repeatability, and reproducibility of the data rely on strictly controlled and validated experimental conditions. The selection of the most relevant biological viscoelastic parameters benefits from standardized modalities of assessments. Attempts were made to derive constants from the experimental data. The Hook constant is obtained in the portion of the curve where a fixed ratio is obtained between load and extension. The Young’s modulus refers to the value of the stress–strain ratio. When skin is stressed by a load, rapid extension takes place at first but then gives way to a region of much less extension. The overall response is nonlinear, although there is a linear portion of the slope. Where extension is directly proportional to the load applied and the material tested will return to its original length, when the load is removed the relationship is said to be ‘‘elastic’’ or ‘‘Hookean.’’ The second phase is typical of an ‘‘elastic’’ material, but the first indicates that the skin becomes ‘‘stiff.’’ The shape of the curve is such that it is very difficult to describe it mathematically. As a residual deformation is commonly present, it interferes with subsequent testing at the same site during the next hours. These changes in mechanical characteristics are sometimes referred to as ‘‘creep,’’ ‘‘viscous extension,’’ or ‘‘viscous slip.’’ Hence, the concept of ‘‘preconditioning procedure’’ is achieved by applying a series of preliminary stresses to the tissue before measuring its mechanical functions. When a series of stress cycles are consecutively applied and removed, slightly different curves are obtained on each occasion. Because of the above considerations, the results of mechanical testing are clearly time dependent. Indeed, the results obtained depend to some extent on the rate at which the stress is applied, the duration for which it is applied, and the previous stress history of the site. While performing in vivo mechanical testing of skin, information is expected in line with other biological parameters. The measurements should provide data consonant with the usual functions of the skin. In fact, in normal and pathological conditions, the relevant mechanical functions of skin represent only a small part of its maximum mechanical capacity. Results unrelated to biological functions of the skin should be disregarded. Such a concept covers several questions to be answered successively when mechanical testing of the skin is to be performed in vivo. ● What is the relevant range of mechanical function in the condition being studied? ● What is the nature of information expected? ● What is the most relevant parameter to be measured? ● What region of the skin is being tested?
● What section of the skin is being tested? ● How do the tissues respond to the forces exerted? ● What is the interpretation to be given in a fourdimensional concept of skin volume and time? In the past, many methods were used to assess the mechanical functions of the skin. They measured different parameters, and the results were hardly comparable because they differed considerably by many qualitative and quantitative aspects. Indeed, the crude data must be interpreted with respect to the method used and the type of test performed. Detailed information about the testing conditions is mandatory. It includes any eventual preconditioning, the orientation, time of application and magnitude of the force exerted, the deformations gained for several load intensities, the body site, and the geometry of the device.
Viscoleastic Function of Facial Skin Using the Suction Method One of the most popular method for measuring skin biomechanics in vivo relies to the so-called suction method [1, 14, 17, 27, 28]. On facial skin, the upper part of the cheeks and the forehead are the sites commonly chosen for the assessments. The Cutometer1 SEM 575 (C + K, Cologne, Germany) is a convenient device equipped with a hand-held probe applied to the skin at constant pressure. The probe has a central suction aperture of 2–6 mm diameter. The diameter of the probe and the intensity and duration of the suction are the critical parameters influencing the results. The accuracy of measurements reaches 0.01 mm in vertical skin extension under stress. Two main operating modalities are possible. One is the fluage test using the time/strain mode (> Fig. 27.1). In this mode, for a given aperture of the probe, the choice of vacuum (from 50 to 500 mbar), the total duration of suction (stress on) and relaxation time (stress off), and the number of measurement cycles are selected. The quantitative parameters describe the elastic deformation and recovery of the skin, the viscoelastic creep after the initial deformation and the initial recovery and the residual deformation. Ue is defined as the immediate elastic distention (ED) corresponding to the steep linear part of the curve computed at a very short interval after application of the suction, usually around 0.1 s. Uv refers to the delayed viscoelastic part of the skin deformation (creep). Uf corresponds to the maximum deformation (MD) combining the elastic distention Ue and the following viscoelastic deformation Uv. Uf is computed after various time intervals ranging from 1 to 10 s. This value
Facial Skin Rheology
. Figure 27.1 Fluage test showing the relationship between the skin extension (E) and time (T). The curve shows the effect of a suction applied for 5 s followed by a release of the suction for 5 s. The immediate elastic distension (ED = Ue) is followed by a delayed viscoelastic phase Uv to reach the maximum distension (MD = Uf). The immediate elastic recovery (ER: Uf – Ur) is followed by a delayed viscoelastic phase ending with a residual distension (RD) following the Ua recovery
of maximum deformation depends on the probe aperture and the applied suction. Ur is defined as the immediate elastic recovery (ER) of the skin after removal of the suction. It is measured in the steep linear part of the recovery, mostly 0.1 s after stopping the suction. Ua is equal to the total recovery deformation of the skin at the endpoint of the recovery phase. RD is the residual deformation of the skin persisting after completion of the stress-off measuring time. All these determinations of parameters are computed by the device according to a predetermined timing of measurement. It is the investigator who makes the choice and there is no standardized procedure as yet. As a result, caution should be taken before comparing data from different studies. From the computed biomechanical parameters, different elastic and viscoelastic ratios have been proposed in order to characterize the mechanical functions of the skin: ● Ua/Uf is defined as the overall (biologic) elasticity. It is expressed in percentage, and it corresponds to the ratio of the total deformation recovery to the total deformation. ● Ur/Ue is defined as the basic elasticity ratio; it is equal to the ratio of the immediate recovery to the immediate deformation without the contribution of the viscoelastic part.
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● Ur/Uf is defined as the relative elastic recovery; it is equal to the ratio of immediate recovery to the total deformation. ● Uv/Ue is defined as the viscoelastic ratio; it is equal to the ratio of the viscoelastic deformation to elastic deformation. Under repetitive measuring cycles, the deformation versus time curves obtained for the second, third, and subsequent deformation cycles are similar to the first, but they progressively shift upwards as a consequence of the residual deformation. The differential distension (DD, m) is calculated as the difference between MDs reached at the last and the first cycles. For example, three to five load (traction) cycles of 2–5-s tractions under negative pressure of 400 mbar are separated by identical relaxation periods (> Fig. 27.2). The second modality test corresponds to the hysteresis procedure using the stress/strain mode. For example, one cycle of progressively increasing suction at a linear rate of 25 mbar/s for 10–20 s is followed by a release of the depression at the same rate (> Fig. 27.3). In this procedure, nonlinear curves are obtained. The suction curve on loading is not superposed by the relaxation curve. During the relaxation period the values of strain do not return to zero and the curve intercepts the strain axis. Hysteresis (HY) represents the area delimited by the traction and relaxation curves given by the stress/strain method. It is measured using image analysis of the graphs yielded by the time/strain method.
Viscoelastic Function of Facial Skin Using the Torque Method The torque method at low stress provides information that may be presented as being similar to that described for the suction method. However, the skin layers subjected to the stress are difficult to identify. It is claimed that the contribution of the stratum corneum to the overall mechanical functions of skin is increased using the torque method. Some equivalence in the mechanical functions of the face and the volar forearm was reported [28]. Such finding await confirmation.
Ultrasound Speed Propagation and Airblown Technique Subtle variations in tensile functions of facial skin is conveniently studied by measuring the speed of propagation
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. Figure 27.2 Repetitive fluage tests showing the effect of preconditioning the skin with a progressive increase in the skin deformation
. Figure 27.3 Hysteresis (HY) procedure showing the evolution of the stress (S) – skin extension (E) under progressive but regular suction until the maximum distension (MD) is gained followed by a progressive relaxation phase. Hysteresis represents the area between the suction and the relaxation curves
corneum suppleness/stiffness influence the data [20, 29]. It is assumed that the velocity of ultrasound propagation is affected by the orientation of the resting tension lines or Langer’s lines [20]. The airblown technique is another ingenious procedure for measuring the skin mechanical functions under low stress [30].
Physiological Changes in Mechanical Functions There is general agreement that age, gender, skin thickness, and location on the body are the four main parameters that influence the rheological functions of the skin. They have to be taken into account before interpreting any given physio-pathological process.
Age Influence
of ultrasound shear waves. The Reviscometer RVM 600 (C + K electronic, Cologne, Germany) is available for that purpose. The resonance running time measurement (RRTM) is inversely correlated with the skin stiffness. Both the dermal mechanical functions and the stratum
Cutaneous aging encompasses distinct features. Chronologic or intrinsic aging depends on genetic factors, lapse of time, and the sum of various effects of diseases and desmotropic drugs (i.e., corticosteroids, phenitoin), as well as physiologic variations and environmental influences with the exception of sun exposure. Photoaging deals with all these features to which chronic exposure
Facial Skin Rheology
to ultraviolet light and near infrared radiations are superimposed [4, 5]. Some authors regard aging of facial skin as a single and direct result of actinic insult, but this opinion may be an oversimplification. In fact, dermatoheliosis is only one aspect of variable importance among subjects of the same age and same phototype. On the face, it is superimposed to both the overall intrinsic aging process mainly responsible for tissue atrophy, and to unrelated opposite hypertrophic changes consisting of compact solar elastosis. In addition, focal hyperplasia develop in the subcutaneous connective tissue where striated muscles are anchored. Such tissue remodeling resulting from distinct origins is responsible for the progressive deepening of the natural expression lines [11]. It is clinically obvious that the mechanical functions of skin are quite different in children and the elderly [1, 7, 13, 18, 22]. They appear correlated with the skin surface patterns and wrinkling [31]. However, measuring them by different test modalities provides controversial findings regarding the nature of the changes and the moment they take place. From the available information, it is probable that the resistance of the dermis to forces exerted parallel to the skin surface increases with age at least until 60 years. Conversely, the vertical resistances at the dermo– epidermal junction, as well as within the dermis and the hypodermis progressively weaken. All age-related changes in skin obviously influence the mechanical functions. Moreover, mechanical stimuli applied to the skin throughout life affect the structure of the cutaneous tissues, which in turn modifies the mechanical functions. Such multiple interrelationships between the various structures of facial skin, the innumerable factors influencing aging, and the complexity of the mechanical properties likely preclude any clear-cut understanding of the problem. Most of the studies on rheological functions of aging skin were focused on the forearms. Little is known about corresponding changes on facial skin. It has been shown that the elevation due to suction depends on the force exerted, the body site, and the area of contact between the probe and the skin. It is particularly difficult to assess the influence of subcutaneous tethering. However, it is likely that the tests using a small hollow probe and producing small elevations of the skin are little influenced by subcutaneous attachments. In these instances, skin extensibility increases while elasticity decreases with aging of facial skin [1]. However, the bulk of elevation experiments reveals wide variations in the deformability of skin in aged individuals. Changes in the elastic rebound of the skin (BE and HY) are more constant, indicating a progressive decrease in these parameters over time.
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Gender Influence The influence of gender on the mechanical functions of skin is a subject of contradictory reports in the literature. Some consider that the skin extensibility is higher, or that the modulus of elasticity is lower, in women than in men, but the reverse opinion has been expressed as well. The difference is in part due to different test modalities.
Skin Thickness Influence The thickness of the dermis is a function of age and gender. It influences the mechanical functions of the skin [1, 32]. This contention holds true when considering the extracellular matrix of the connective tissue. However, facial skin thickness is considerably influenced by the size of the sebaceous glands. Such a double-component structure exhibits peculiar biomechanical functions. The importance of the facial skin thickness is further complicated by the variable extent in solar elastosis.
Credentiating Anti-aging Treatments During the past decades, the dermocosmetic science applied to facial skin has considerably influenced the clinical presentation of aging. Specific cosmetics, cosmeceuticals, and drugs are designed for corrective purposes. A few studies have been conducted to assess the rheological changes following the regular use of specific topical treatments [24, 33, 34]. The effects were correlated with the histological nature of wrinkles and improvement of the skin relief. Other minimally invasive methods are available for improving the appearance of aging face [35]. Among them, the filling procedures, the peelings, the botulinum toxin, the photorejuvenation, and still other nonablative resurfacing procedures are very popular. No information is available regarding the induced changes in mechanical functions of skin. The same lack of information applies to the effects of skin lifting, the lipoaugmentation, and other invasive surgical procedures. Active cosmetic products, called cosmeceuticals in some countries and quasi-drugs in others are rapidly expanding and becoming increasingly sophisticated. The potential value of these formulations for skin health is undisputable. Yet, both the consumer and the dermocosmetologist are challenged while evaluating their benefits. Many prescription dermatologics fall short of patient
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expectations, opening the way for the use of cosmeceuticals to enhance the outcome. However, the physician has some guarantee that the pharmaceuticals are at least moderately effective. When dealing with facial skin, testing the effects of products on the rheological functions of skin is difficult to predict when using the suction method on the model of the forearm skin. Only indirect and partial information is obtained. By contrast, the torque method has been reported to give equivalent information on the face and the forearms [28].
Conclusion The evaluation of the mechanical functions of the skin is useful in the dermocosmetic field of investigation. A series of different methods using dedicated devices provide different information, although those useful in practice are limited in number. The lack of standardization often precludes the comparison of results obtained by different groups of workers. In terms of bioengineering, the skin withstands and transmits mechanical forces through specific deformations. Creep and stress relaxation effects are well recognized. This fact implies that the time rate of application of forces onto the skin also influences the data while assessing the load transmitting capabilities of the tissue. The comparison between different rheological methods applicable to the face indicates that the progressive increase and release of suction in the stress/strain mode yields the greatest relative variations with age. Hence this methodological approach appears well suited to study facial skin aging and the efficacy of products aiming at its correction. Tests performed in vivo are likely to have the most value for clinical purposes, but have special problems of their own. ● The dermis in vivo is under a variable degree of resting tension. Langer’s lines are an expression of the state of resting tension in the skin and indicate the orientation of maximum tension. ● There are intimate connections between the dermis, the epidermis, and its pilosebaceous adnexae, as well as between the dermis and the hypodermis. It is virtually impossible to isolate the dermis from its intimately associated neighboring structures when tests are performed in vivo. Inevitably, the test performed are, in part, also testing the epidermal, adnexal, and hypodermal functions. It is, however, quite evident that in
most cases the results mainly reflect the properties and functions of the dermal collagen fiber bundles. The scope of these tests must be borne in mind while interpreting their results. ● Clearly, the results of mechanical testing depend on the dimensions of the site being investigated. It is relatively easy to define the length and breadth of the area tested, but the skin thickness decreases with age and is greater in men than women. It is susceptible to endocrine and environmental influences. In addition, the mechanical functions of the dermis are time dependent.
References 1. Pie´rard GE. EEMCO guidance to the in vivo assessment of tensile functional properties of the skin. Part 1: relevance to the structures and ageing of the skin and subcutaneous tissues. Skin Pharmacol Appl Skin Physiol. 1999;12:352–362. 2. Hendriks FM, Brokken D, Oomens CW, Bader DL, Baaijens FP. The relative contributions of different skin layers to the mechanical behaviour of human skin in vivo using suction experiments. Med Eng Phys. 2006;28:259–266. 3. Ryu HS, Joo HY, Kim SO, Park KC, Youn SW. Influence of age and regional differences on skin elasticity as measured by the Cutometer1. Skin Res Technol. 2008;14:354–358. 4. Schroeder P, Haendeler J, Krutmann J. The role of near infrared radiation in photoaging of the skin. Exp Gerontol. 2008;43:629–632. 5. Farage MA, Miller KW, Elsner P, Maibach HI. Intrinsic and extrinsic factors in skin ageing: a review. Int J Cosmet Sci. 2008;30:87–95. 6. Ambroisine L, Ezzedine K, Elfakir A, Gardinier S, Latreille J, Mauger E, et al. Relationships between visual and tactile features and biophysical parameters in human facial skin. Skin Res Technol. 2007;13:176–183. 7. Pie´rard-Franchimont C, Paquet P, Quatresooz P, Pie´rard GE. Smoothing the mosaic subclinical melanoderma by calcipotriol. J Eur Acad Dermatol Venereol. 2007;21:657–661. 8. Akazaki S, Nakagawa H, Kazama H, Osanai O, Kawai M, Takema Y, et al. Age-related changes in skin wrinkles assessed by a novel three-dimensional morphometric analysis. Br J Dermatol. 2002;147:689–695. 9. Batisse D, Bazin R, Baldeweck T, Querleux B, Le´veˆque JL. Influence of age on the wrinkling capacities of skin. Skin Res Technol. 2002;8:148–154. 10. Pie´rard GE, Uhoda I, Pie´rard-Franchimont, C. From skin microrelief to wrinkles. An area ripe for investigation. J Cosmet Dermatol. 2003;2:21–28. 11. Quatresooz P, Thirion L, Pie´rard-Franchimont C, Pie´rard GE. The riddle of genuine skin microrelief and wrinkles. Int J Cosmet Sci. 2006;28:389–395. 12. Fukuda Y, Fujimura T, Moriwaski S, Kitahara T. A new method to evaluate lower eyelid sag using three-dimensional image analysis. Int J Cosmet Sci. 2005;27:283–290. 13. Saito N, Nishijima T, Fujimura T, Moriwaki S, Takema Y. Development of a new evaluation method for cheek sagging using a Moire 3D analysis system. Skin Res Technol. 2008;14:287–292.
Facial Skin Rheology 14. Pie´rard GE, Henry F, Castelli D, Ries G. Ageing and rheological properties of facial skin in women. Gerontology. 1998;44:159–161. 15. Weiss RA, McDaniel DH, Geronemus RG, Weiss MA, Beasley KL, Munavalli GM, et al. Clinical trial of a novel non-thermal LED array for reversal of photoaging: clinical, histologic, and surface profilometric results. Lasers Surg Med. 2005;36:85–91. 16. Staloff IA, Guan E, Katz S, Rafailovitch M, Sokolov A, Sokolov S. An in vivo study of the mechanical properties of facial skin and influence of aging using digital image speckle correlation. Skin Res Technol. 2008;14:127–134. 17. Pie´rard-Franchimont C, Cornil F, Dehavay J, Deleixhe-Mauhin F, Letot B, Pie´rard GE. Climacteric skin ageing of the face. A prospective longitudinal intent-to-treat trial on the effect of oral hormone replacement therapy. Maturitas. 1999;32:87–93. 18. Pie´rard GE, Kort R, Letawe C, Olemans C, Pie´rard-Franchimont C. Biomechanical assessment of photodamage. Derivation of a cutaneous extrinsic ageing score. Skin Res Technol. 1995;1:17–20. 19. Pie´rard GE, Lapie`re ChM. Microanatomy of the dermis in relation to relaxed skin tension lines and Langer’s lines. Am J Dermatopathol. 1987;9:219–224. 20. Hermanns-Leˆ T, Jonlet F, Scheen A, Pie´rard GE. Age – and body mass index-related changes in cutaneous shear wave velocity. Exp Gerontol. 2001;36:363–372. 21. Jacquet E, Josse G, Khatyr F, Garcin C. A new experimental method for measuring skin’s natural tension. Skin Res Technol. 2008;14:1–7. 22. Takema Y, Yorimoto Y, Kawai M, Imokawa G. Age-related changes in the elastic properties and thickness of human facial skin. Br J Dermatol. 1994;131:641–648. 23. Hermanns-Leˆ T, Uhoda I, Smitz S, Pie´rard GE. Skin tensile properties revisited during ageing. Where now, where next? J Cosmet Dermatol. 2004;3:35–40. 24. Pie´rard-Franchimont C, Castelli D, Van Cromphaut I, Bertin C, Ries G, Cauwenbergh G, et al. Tensile properties and contours of aging facial skin. A controlled double-blind comparative study of the effects of retinol, melibiose-lactose and their association. Skin Res Technol. 1998;4:237–243.
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25. Khatyr F, Imberdis C, Vescovo P, Varchon D, Lagarde JM. Model of the viscoelastic behaviour of skin in vivo and study of anisotropy. Skin Res Technol. 2004;10:96–103. 26. Delalleau A, Josse G, Lagarde JM, Zahouani H, Bergheau JM. A nonlinear elastic behavior to identify the mechanical parameters of human skin in vivo. Skin Res Technol. 2008;14:152–164. 27. Khatyr F, Imberdis C, Varchon D, Lagarde JM, Josse G. Measurement of the mechanical properties of the skin using the suction test. Skin Res Technol. 2006;2:24–31. 28. Bazin R, Fanchon C. Equivalence of face and volar forearm for the testing of moisturizing and firming effect of cosmetics in hydration and biomechanical studies. Int Cosmet Sci. 2006;28:453–460. 29. Xhauflaire-Uhoda E, Fontaine K, Pie´rard GE. Kinetics of moisturizing and firming effects of cosmetic formulations. Int J Cosmet Sci. 2008;30:131–138. 30. Fujimura T, Osanai O, Moriwaki S, Akazaki S, Takema Y. Development of a novel method to measure the elastic properties of skin including subcutaneous tissue: new age-related parameters and scope of application. Skin Res Technol. 2008;14:504–511. 31. Ahn S, Kim S, Lee H, Moon S, Chang I, et al. Correlation between a Cutometer and quantitative evaluation using Moire topography in age-related skin elasticity. Skin Res Technol. 2007;13:280–284. 32. Smalls LK, Wickett RR, Visscher MO. Effect of dermal thickness, tissue composition, and body site on skin biomechanical properties. Skin Res Technol. 2006;12:43–49. 33. Pie´rard GE, Henry F, Pie´rard-Franchimont C. Comparative effect of short-time topical tretinoin and glycolic acid on mechanical properties of photodamaged facial skin in HRT-treated menopausal women. Maturitas. 1996;23:273–277. 34. Uhoda I, Faska N, Robert C, Cauwenbergh G, Pie´rard GE. Split face study of the cutaneous tensile effect of a 2-dimethylaminoethanol (deanol) gel. Skin Res Technol. 2002;8:164–167. 35. Bogle MA. Minimally invasive techniques for improving the appearance of the aging face. Expert Rev Dermatol. 2007;2:427–435.
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32 Fibulin-5 Deposition in Human Skin: Decrease with Aging and UVB Exposure and Increase in Solar Elastosis Satoshi Amano
Introduction Skin aging is classified into two types, intrinsic aging and photoaging. Intrinsic aging is a basic biological process common to all living things, and can be characterized as age-dependent deterioration of skin functions and structures, such as epidermal atrophy and epidermal–dermal junction flattening [1]. Histologically, intrinsically aged skin has an atrophied extracellular matrix with a reduced amount of elastin [2]. On the other hand, photoaging is well known to be a consequence of chronic exposure to sunlight. Sun-exposed skin, such as the skin on the face or neck, is apparently prematurely aged compared with the relatively sun-protected skin of the trunk, and is characterized by various clinical features, including wrinkles, sagging, roughness, sallowness, pigmentary changes, telangiectasia, and neoplasia [3, 4], and histological features of sun-exposed skin including cellular atypia, loss of polarity, epidermal–dermal junction flattening, a decrease in collagen, and dermal elastosis, with abnormal deposition of elastotic material in the dermis [1, 5]. Damage to skin collagen and elastin (extracellular matrix) is the hallmark of long-term exposure to solar ultraviolet irradiation, and is believed to be responsible for the wrinkled appearance of sun-exposed skin [5]. The fibulin gene family comprises five distinct genes that encode more than eight protein products via alternative splicing [6]. Fibulins are widely expressed secreted proteins found in the blood and in the basement membranes and stroma of most tissues, where they self-associate [7, 8], and/or interact with a variety of extracellular matrix components, including fibronectin, laminin, nidogen, aggrecan, versican, endostatin, fibrillin, and elastin [6, 9–11]. Fibulins are thought to be involved in the assembly and stabilization of extracellular matrix structures, and have also been implicated in regulating organogenesis, vasculogenesis, fibrogenesis, and tumorigenesis [12–14].
The newest member of the fibulin family is fibulin-5 (known as EVEC [15] or DANCE [16]), a 448-amino acid glycoprotein with interesting structural features; it contains an integrin-binding RGD motif, six calcium-binding epidermal growth factor-like repeats, a Pro-rich insert in the first calcium-binding epidermal growth factor-like repeat, and a globular C-terminal domain [15, 16]. Functionally, fibulin-5 binds avb3, avb3, and a9b1 integrins [10], and mediates endothelial cell adhesion via its RGD motif [16]. Inactivation of the fibulin-5 gene in mice, produces profound elastinopathy in the skin, lung, and vasculature [10, 11]. In humans, mutations in fibulin-5 have been found to cause cutis laxa [17]. Elastic fibers are composed of an amorphous elastin core surrounded by a peripheral mantle of microfibrils. Soluble tropoelastin monomers are polymerized and crosslinked to form insoluble elastin, which is essential for the assembly of elastic fibers [18]. However, self-association of tropoelastin monomers alone is not sufficient to form elastic fibers indicating the need for other processes or substances. Microfibrils are 10–12 nm filaments in the extracellular matrices, and composed of many proteins such as fibrillin-1 and -2 [19–21], microfibril-associated glycoproteins (MAGPs) [22, 23], and latent transforming growth factor b-binding proteins (LTBPs) [24, 25]. Microfibrils are considered to provide a scaffold for the polymerization of elastin, and play an essential role in elastogenesis. The aim of this study was to explore the changes of elastic fibers during the skin aging process. Fibulin-5 expression in both normal and actinically damaged skin when compared with expression of elastin, fibulin-2, and fibrillin-1 showed that fibulin-5 decreased in an age-dependent manner in the reticular dermis, and the reduction was enhanced by UVB irradiation. Moreover, acute UVB irradiation markedly reduced fibulin-5. However, fibulin-5 was also found to accumulate in solar
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_32, # Springer-Verlag Berlin Heidelberg 2010
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elastosis, together with other elastic fiber components, such as elastin, fibulin-2, and fibrillin-1. The results indicated that fibulin-5 is an early marker of skin aging, and that the early loss of fibulin-5 in the dermis may prefigure the age-dependent reduction of other elastic fiber components.
Localization of Fibulin-5 in the Dermis of Young Human Sun-Protected Skin In the reticular dermis of young sun-protected skin from the upper arm of a 17-year-old female, fibulin-5 was colocalized with other elastic fiber components, such as elastin, fibrillin-1, and fibulin-2 (> Fig. 32.1a–d). In contrast with fibrillin-1, fibrillin-2 was not detected in skin from children and adults (data not shown). In the papillary dermis, fibulin-5 showed candelabra-like structures perpendicular to the epidermis, resembling those of the other elastic fiber components (> Fig. 32.1a–d). In areas just beneath the epidermis, fibulin-5 does not go up to the dermal–epidermal junction like fibrillin-1 which was observed there as fiber structures that may be inserted into the epidermal basement membrane [26, 27] (> Fig. 32.1b, c). The fiber structure of fibulin-2 was not as sharp in the papillary dermis (> Fig. 32.1a) as those of elastin, fibrillin-1, or fibulin-5 (> Fig. 32.1a–c), although it was clearer in the reticular dermis (> Fig. 32.1d).
Age-Dependent Changes of Fibulin-5 Distribution in the Dermis of SunProtected or Sun-Exposed Skin In the dermis of sun-protected thigh skin from 5-, 13-, and 16-year-old subjects, fibulin-5 showed candelabralike structures in the papillary dermis, and was associated with elastic fibers composed of other elastic fiber components, such as elastin, fibrillin-1, and fibulin-2 (> Fig. 32.2a–l) in the reticular dermis. However, the staining intensity of fibulin-5 (> Fig. 32.2n) was reduced as compared with that of the other elastic fiber components (> Fig. 32.2m, o, and p) in the reticular dermis of skin from a 34-year-old subject. Fibulin-5 (> Fig. 32.2r) was almost absent in the reticular dermis of skin from a 75-year-old women. Similarly, fibulin-5 associated with elastic fibers was reduced in the reticular dermis of sun-protected upper arm skin from a 36-year-old subject as compared with those from 11-, 17-, and 24-year-old subjects (data not shown). Moreover, fibulin-5 associated
with elastic fibers was markedly reduced as compared with fibrillin-1 and fibulin-2 in the reticular dermis of abdomen skin from 34- and 75-year-old subjects (data not shown). On the other hand, in the papillary dermis, fibulin-5 maintained its staining intensity, although the number of stained fibers seemed to be reduced with age (> Fig. 32.2n, r), whereas elastin was age-dependently reduced much more markedly in papillary dermis than in reticular dermis (> Fig. 32.2m, q). In sun-exposed skin, fibulin-5 was mostly lost in the dermis of cheek skin even from 20- and 45-year-old subjects (> Fig. 32.3b, f ), and this change occurred much earlier than that in sun-protected skin (> Fig. 32.2). Elastic fiber structures in the dermis of 45-year-old skin (> Fig. 32.3e, g, and h) appeared to be thicker than those of 20-year-old skin (> Fig. 32.3a, c, and d), and was intermediate in pattern between the 20-year-old skin, and the elastic fibers observed in the dermis of a 76-year-old subject (> Fig. 32.3i, k, and j), suggesting that the 45-year-old skin may be progressing to solar elastosis. However, while increased deposition of fibulin-5 was observed in solar elastosis (> Fig. 32.3j), as was observed for other elastic fiber components, fibulin-5 decreased with age in sun-exposed skin before solar elastosis appeared (> Fig. 32.3b, f ). Fibulin-5-deficient mice were reported to develop marked elastinopathy owing to the disorganization of elastic fibers, resulting in loose skin, vascular abnormalities, and emphysematous lung [10, 11]. Since fibulin-5 has an integrin-binding N-terminal domain, fibulin-5 is thought to stabilize the attachment of cells to elastic fibers, and to contribute to the organization of elastic fibers [10]. Recently, fibulin-5 is reported to be a key protein for the induction of elastic fiber formation and full intact form of fibulin-5 diminishes with age [28]. Therefore the loss of fibulin-5 may decrease the stability of elastic fibers by disturbing the interactions between dermal cells and elastic fibers or among elastic fiber components, and may contribute to the atrophy of elastic fibers during aging. The hallmark of actinic damage of the skin changes is associated with deposition of elastotic materials in the dermis [1]. Previous immunohistochemical studies reported an increased deposition of elastin, versican, hyaluronic acid, fibrillin, and fibulin-2 in areas of solar elastosis [29–31]. Fibulin-5 deposition also increases in solar elastosis. The mechanism of the increase in the expression of these elastic fiber components, leading to abnormal deposition in the dermis of actinically damaged skin, remains unknown. However, since all elastic fiber components, including fibulin-5, are increased in solar
Fibulin-5 Deposition in Human Skin: Decrease with Aging and UVB Exposure and Increase in Solar Elastosis
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. Figure 32.1 Localization of elastin, fibulin-5, fibrillin-1, and fibulin-2 in the dermis of young human sun-protected skin. The localization of elastin (a and e), fibulin-5 (b and f), fibrillin-1 (c and g) or fibulin-2 (d and h) in sun-protected skin from upper arm of a 17-year-old female was examined by means of immunohistochemistry using specific antibodies
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. Figure 32.2 Age-dependent changes of expression of elastin, fibulin-5, fibrillin-1, and fibulin-2 in the dermis of sun-protected skins. The localization of elastin (a, e, i, m, and q), fibulin-5 (b, f, j, n, and r), fibrillin-1 (c, g, k, o, and s) or fibulin-2 (d, h, l, p, and t) in the dermis of sun-protected thigh skin was examined by means of immunohistochemistry using specific antibodies. Age-dependent changes of the elastic fiber components were examined in the dermis of skins from subjects in the age range from 5 to 75 year old. It should be noted that fibulin-5 decreased especially markedly in the reticular dermis with aging (Image reproduced, with permission, from Kadoya et al. [35]
elastosis, the mechanism may activate the elastic fiber developmental program. Since fibulin-5 was observed to be extremely reduced in the aging dermis, it is possible that the control of gene expression, protein synthesis, or
deposition of fibulin-5 may be different from those of other elastic fiber components. Further studies are needed to clarify the role of fibulin-5 in normal aging and in the pathogenesis of solar elastosis.
Fibulin-5 Deposition in Human Skin: Decrease with Aging and UVB Exposure and Increase in Solar Elastosis
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. Figure 32.3 Age-dependent changes of expression of elastin, fibulin-5, fibrillin-1, and fibulin-2 in the dermis of sun-exposed skin. The localization of elastin (a, e, and i), fibulin-5 (b, f, and j), fibrillin-1 (c, g, and k) or fibulin-2 (d, h, and l) in the dermis of sun-exposed cheek skins was examined by means of immunohistochemistry using specific antibodies, as described in Materials and Methods. Age-dependent changes of the elastic fiber components were examined in the dermis of skin from subjects in the age range from 20 to 76 year old. It should be noted that the reduction of fibulin-5 in the reticular dermis occurred earlier in sun-exposed skin than in sun-protected skin. Increased deposition of fibulin-5, as well as the other elastic fiber components, was observed in solar elastosis (Image reproduced, with permission, from Kadoya et al. [35]
Reduction of Fibulin-5 in the Dermis After UVB Irradiation Since fibulin-5 in the dermis was reduced in sun-exposed skin earlier than that in sun-protected skin, the effect of UVB irradiation on the fibulin-5 distribution in buttock skin from two male volunteers was explored. A single UVB irradiation at 2 MED decreased fibulin-5 (> Fig. 32.4i, j), fibulin-2 (> Fig. 32.4k, l ), and elastin (> Fig. 32.4g, h) levels in the dermis markedly, moderately, and weakly, respectively, as compared with those in non treated skin (> Fig. 32.4a–f ). Fibulin-5 deposition decreased much earlier in sunexposed skins than in sun-protected skins. Furthermore, UVB-irradiation induced the degradation of fibulin-5 deposited in the dermis. Matrix-degrading metalloproteinase messenger RNAs, proteins and activities are known to be induced in human skin in vivo within hours of
exposure to UVB irradiation, and may degrade collagen and elastin in skin [32]. Fibulin-2 was reported to be degraded by matrix metalloproteinases (stromelysin, matrilysin), circulating proteases (thrombin, plasmin, kallikrein), leucocyte elastase, and mast cell chymase [33]. Smaller degradation products of fibulin-2 were also detected in actinic elastosis, presumably reflecting increased proteinase activity in photodamaged skin [31]. The sensitivity of fibulin-5 to proteinases has not yet been reported, but proteinases induced by the exposure of skin to UV may be involved in the early loss of fibulin-5 observed in the dermis.
Conclusion Fibulin-5 content in the reticular dermis decreases with age, and decreases earlier than other elastic fiber
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. Figure 32.4 Reduction of fibulin-5 in the dermis after UVB irradiation. The localization of elastin (a, b, g, and h), fibulin-5 (c, d, I, and j), or fibulin-2 (e, f, k, and i) in the dermis of buttock skin was compared between non treated sites (a–f) and UVB-irradiated sites (g–l) by means of immunohistochemistry using specific antibodies. It should be noted that fibulin-5 was markedly reduced in the dermis by UVB-irradiation (Image reproduced, with permission, from Br J Dermatol. 2005;153(3):607–612)
components, such as elastin, fibrillin-1, and fibulin-2. The reduction of fibulin-5 was enhanced by UVB exposure, and occurred in sun-exposed skin much earlier than in sun-protected skin. Therefore, UVB is likely to be one of the major factors causing impairment of elastic fibers during aging, and the early loss of fibulin-5 may signal
the later changes of elastic fibers during aging, especially photoaging. Therefore, fibulin-5 is proposed to be a good marker of skin aging, especially photoaging. Interestingly, fibulin-5 deposition is enhanced in solar elastosis, suggesting that solar elastosis involves the global activation of genes for elastic fiber components.
Fibulin-5 Deposition in Human Skin: Decrease with Aging and UVB Exposure and Increase in Solar Elastosis
Fibulin-5-overexpressing cells enhanced the assembly of elastic fibers in cultured normal human dermal fibroblasts, suggesting that fibulin-5 was an important microfibril constituent for the assembly of elastic fibers [34]. Thus, fibulin-5 may be a potential target to prevent or delay the deterioration of elastic fibers during the skinaging process in human.
Cross-references > DNA
Damage and Repair in Skin Aging
References 1. Lavker RM. Structural alterations in exposed and unexposed aged skin. J Invest Dermatol. 1979;73:59–66. 2. Braverman IM, Fonferko E. Studies in cutaneous aging: I. The elastic fiber network. J Invest Dermatol. 1982;78:434–443. 3. Gilchrest BA. Skin aging and photoaging: an overview. J Am Acad Dermatol. 1989;21:610–613. 4. Griffiths CE. The clinical identification and quantification of photodamage. Br J Dermatol. 1992;127:37–42. 5. Kligman AM, Grove GL, Hirose R, et al. Topical tretinoin for photoaged skin. J Am Acad Dermatol. 1986;15:836–859. 6. Timpl R, Sasaki T, Kostka G, et al. Fibulins: a versatile family of extracellular matrix proteins. Nat Rev Mol Cell Biol. 2003;4:479–489. 7. Balbona K, Tran H, Godyna S, et al. Fibulin binds to itself and to the carboxyl-terminal heparin-binding region of fibronectin. J Biol Chem. 1992;267:20120–20125. 8. Pan TC, Sasaki T, Zhang RZ, et al. Structure and expression of fibulin-2, a novel extracellular matrix protein with multiple EGFlike repeats and consensus motifs for calcium binding. J Cell Biol. 1993;123:1269–1277. 9. Aspberg A, Adam S, Kostka G, et al. Fibulin-1 is a ligand for the C-type lectin domains of aggrecan and versican. J Biol Chem. 1999;274:20444–20449. 10. Nakamura T, Lozano PR, Ikeda Y, et al. Fibulin-5/DANCE is essential for elastogenesis in vivo. Nature. 2002;415:171–175. 11. Yanagisawa H, Davis EC, Starcher BC, et al. Fibulin-5 is an elastinbinding protein essential for elastic fibre development in vivo. Nature. 2002;415:168–171. 12. Zhang HY, Timpl R, Sasaki T, et al. Fibulin-1 and fibulin-2 expression during organogenesis in the developing mouse embryo. Dev Dyn. 1996;205:348–364. 13. Tran H, Tanaka A, Litvinovich SV, et al. The interaction of fibulin-1 with fibrinogen. A potential role in hemostasis and thrombosis. J Biol Chem. 1995;270:19458–19464. 14. Roark EF, Keene DR, Haudenschild CC, et al. The association of human fibulin-1 with elastic fibers: an immunohistological, ultrastructural, and RNA study. J Histochem Cytochem. 1995; 43:401–411. 15. Kowal RC, Richardson JA, Miano JM, et al. EVEC, a novel epidermal growth factor-like repeat-containing protein upregulated in embryonic and diseased adult vasculature. Circ Res. 1999;84:1166–1176. 16. Nakamura T, Ruiz-Lozano P, Lindner V, et al. DANCE, a novel secreted RGD protein expressed in developing, atherosclerotic, and balloon-injured arteries. J Biol Chem. 1999;274:22476–22483.
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17. Loeys B, Van Maldergem L, Mortier G, et al. Homozygosity for a missense mutation in fibulin-5 (FBLN5) results in a severe form of cutis laxa. Hum Mol Genet. 2002;11:2113–2118. 18. Urry DW. Entropic elastic processes in protein mechanisms. II. Simple (passive) and coupled (active) development of elastic forces. J Protein Chem. 1988;7:81–114. 19. Sakai LY, Keene DR, Engvall E. Fibrillin, a new 350-kD glycoprotein, is a component of extracellular microfibrils. J Cell Biol. 1986;103:2499–2509. 20. Zhang H, Hu W, Ramirez F. Developmental expression of fibrillin genes suggests heterogeneity of extracellular microfibrils. J Cell Biol. 1995;129:1165–1176. 21. Zhang H, Apfelroth SD, Hu W, et al. Structure and expression of fibrillin-2, a novel microfibrillar component preferentially located in elastic matrices. J Cell Biol. 1994;124:855–863. 22. Gibson MA, Hughes JL, Fanning JC, et al. The major antigen of elastin-associated microfibrils is a 31-kDa glycoprotein. J Biol Chem. 1986;261:11429–11436. 23. Gibson MA, Hatzinikolas G, Kumaratilake JS, et al. Further characterization of proteins associated with elastic fiber microfibrils including the molecular cloning of MAGP-2 (MP25). J Biol Chem. 1996;271:1096–1103. 24. Gibson MA, Hatzinikolas G, Davis EC, et al. Bovine latent transforming growth factor beta 1-binding protein 2: molecular cloning, identification of tissue isoforms, and immunolocalization to elastinassociated microfibrils. Mol Cell Biol. 1995;15:6932–6942. 25. Taipale J, Lohi J, Saarinen J, et al. Human mast cell chymase and leukocyte elastase release latent transforming growth factor-beta 1 from the extracellular matrix of cultured human epithelial and endothelial cells. J Biol Chem. 1995;270:4689–4696. 26. McGrath JA, Ishida-Yamamoto A, Shimizu H, et al. Immunoelectron microscopy of skin basement membrane zone antigens: a preembedding method using 1-nm immunogold with silver enhancement. Acta Derm Venereol. 1994;74:197–200. 27. Haynes SL, Shuttleworth CA, Kielty CM. Keratinocytes express fibrillin and assemble microfibrils: implications for dermal matrix organization. Br J Dermatol. 1997;137:17–23. 28. Hirai M, Ohbayashi T, Horiguchi M, et al. Fibulin-5/DANCE has an elastogenic organizer activity that is abrogated by proteolytic cleavage in vivo. J Cell Biol. 2007;176:1061–1071. 29. Bernstein EF, Fisher LW, Li K, et al. Differential expression of the versican and decorin genes in photoaged and sun-protected skin. Comparison by immunohistochemical and northern analyses. Lab Invest. 1995;72:662–669. 30. Bernstein EF, Underhill CB, Hahn PJ, et al. Chronic sun exposure alters both the content and distribution of dermal glycosaminoglycans. Br J Dermatol. 1996;135:255–262. 31. Hunzelmann N, Nischt R, Brenneisen P, et al. Increased deposition of fibulin-2 in solar elastosis and its colocalization with elastic fibres. Br J Dermatol. 2001;145:217–222. 32. Fisher GJ, Datta SC, Talwar HS, et al. Molecular basis of sun-induced premature skin ageing and retinoid antagonism. Nature. 1996;379: 335–339. 33. Sasaki T, Mann K, Murphy G, et al. Different susceptibilities of fibulin-1 and fibulin-2 to cleavage by matrix metalloproteinases and other tissue proteases. Eur J Biochem. 1996;240:427–434. 34. Katsuta Y, Ogura Y, Iriyama S, et al. Fibulin-5 accelerates elastic fibre assembly in human skin fibroblasts. Exp Dermatol. 2008;17:837–842. 35. Kadoya K, Sasaki T, Kostka G, Timpl R, Matsuzaki K, Kumagai N, Sakai LY, Nishiyama T, Amano S. Fibulin-5 deposition in human skin: decrease with ageing and ultraviolet B exposure and increase in solar elastosis. Br J Dermatol. 2005;153:607–612.
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43 Global Warming and its Dermatologic Impact on Aging Skin Young Hui . Haw-Yueh Thong . Howard I. Maibach
Introduction The large-scale impacts of global warming are widely debated in both mainstream and academic literatures [1], but the specific consequences of global warming for individual scientific disciplines are poorly discussed. Many scientific disciplines are yet to plan for global warming; not only for changing temperature gradations, but also for the effects on plant, animal, and man. It is suspected that global climate change will alter skin – in ways not yet determined. Little research has been done to assess the challenges that dermatology might face in the future in the light of global warming; much less research has been done in the area of skin care for old people, but such studies can nevertheless be extrapolated from some upcoming trends.
Ultraviolet Effects Decades of ozone depletion have focused scrutiny on the effects of increased ultraviolet (UV) exposure. While not a direct consequence of global warming, increased UV exposure may result from factors such as cloud cover and aerosols [2]. Warmer temperatures also appear to increase the incidence of skin cancer from UV. Studies suggest that a rise of just 2 C may increase the rates of such cancer by up to 10% [3]. As people retire and temperatures rise, more may seek recreation outdoors. Not only the intensity, but also the duration of UV that people experience will potentially increase. Beyond the increase in UV penetration, such changes in social habits may be the greatest factor in future skin cancer rates [4, 5].
The Hot Zone: Skin Infections A shift in temperature would change the dynamics of vector-borne diseases, such as malaria and mosquitoes or leishmaniasis and sand flies [6–9]. Cases of leishmaniasis,
in particular, increased during the warming phase of El Nino and decreased during the cooling phase of La Nina in Columbia [10]. Tick-borne diseases, susceptible to climate change, have also increased [11]. But current data cannot prove a causal relationship for either of these cases. To give a convincing proof one requires more detailed and direct evidence of climate change impacts. The impact of global warming on pathogen outbreaks appears more definite. The Monteverde harlequin frog was driven to extinction by outbreaks of the pathogenic chytrid fungus Batrachochytrium, which occurred when highland locales warmed [12]. Similar outbreaks among humans have not yet been witnessed, but the increased proclivity of Staphylococcus, Streptococcus, and enteric bacteria to colonize humans in warmer climes has been observed [13, 14]. Other research has also suggested that increased temperature and humidity generally favor bacterial growth [15]. Such challenges await dermatologists as humans venture into the fires of global warming. Pathogen outbreaks, with inherent potential for antimicrobial resistance, will stare at dermatologists if global warming proceeds. Those who are most at risk, such as the elderly or infirm, should be advised most of all.
Worsening Weather for Dermatologists Global warming may change the patterns of severe weather events. These changes would generate wide effects upon public health and disease transmission. This broader transmission can only be detrimental to a vulnerable, aging society. If global warming will bring more extreme weather in the future, it may be informative to examine recent weather for indications as to the future trends in the weather. Hurricane Katrina brought a deluge of skin infection reports. The Centers for Disease Control and Prevention (CDC) highlighted methicillin-resistant Staphylococcus aureus (MRSA), Vibrio vulnificus, and V. parahaemolyticus infections among the rescued, and tinea corporis, folliculitis, miliaria, and arthropod bites among rescuers [16].
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Septic shock was reported among victims with V. vulnificus [17]. The Indian Ocean tsunami of December 2004 is another case to consider. Although the cause of this calamity may not be global warming, it still carries consequences similar to any massive precipitation event. A sampling of survivors showed 515 (66.3%) of them suffering from skin or soft-tissue infection [18]. Flooding contaminated freshwater with a variety of exotic pathogens, as well as multidrug-resistant bacteria and polymicrobial infections [19–21]. Such challenges are faced by all rescue efforts and doctors should familiarize themselves with those challenges if global warming leads to more occasions for rescue work.
Conclusion The peril of global warming, if it comes to pass, is dangerously clear. It is evident why the reduction of greenhouse gases has become a top social and political priority. Its impact on the biodiversity of the Earth may be tremendous; some predict over a million extinctions of animal and plant species by the middle of this century [22]. While in all likelihood humanity will not be among them, the potential toll on humanity remains immense. Health professionals should be prepared for worst-case scenarios; thus, it is imperative for all, including dermatologists, to investigate and ready themselves for likely future challenges. At this time, what is needed to act is knowledge of what will be faced. Taken together, one or more global-oriented research institutes dedicated to understanding global warming’s skin-related issues should be initiated to define the extent of the problem and create prophylactic and therapeutic interventions. Time does not wait; so action has to begin now.
Cross-references > Effect
of Ozone on Cutaneous Tissues Vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model > Skin Photodamage Prevention: State of the Art and New Prospects > In
References 1. Flannery T. The Weather Makers: How Man is Changing the Climate and What It Means for Life on Earth. New York: Atlantic Monthly Press, 2006.
2. Kerr JB, Seckmeyer G. Surface ultraviolet radiation: past and future. In: Scientific Assessment of Ozone Depletion, Geneva: Global Ozone Research and Monitoring Project, 2002. 3. van der Leun JC, de Gruijl FR. Climate change and skin cancer. Photochem Photobiol Biol Sci. 2002;1:324–326. 4. Diffey BL. Human exposure to ultraviolet radiation. In: Hawk JLM (ed) Photodermatology. London: Arnold, 1999, pp. 5–24. 5. Diffey BL. Climate change, ozone depletion and the impact of ultraviolet exposure on human skin. Phys Med Biol. 2004;49: R1–R11. 6. Kolodynski J, Malinowska A. Impacts of climate change on infectious diseases. Wiad Parazytol. 2002;48:29–37. 7. Kovats RS, Campbell-Lendrum DH. McMichael AJ, et al. Early effects of climate change: do they include changes in vector-borne disease. Philos Trans R Soc Lond B Biol Sci. 2001;356:1057–1068. 8. Bormane A, Lucenko I, Duks A, et al. Vectors of tickborne diseases and epidemiological situation in Latvia in 1993–2002. Int J Med Microbiol. 2004;293(Suppl 37): 36–47. 9. Sutherst RW. Global change and human vulnerability to vectorborne diseases. Clin Microbiol Rev. 2004;17:136–173. 10. Cardenas R, Sandoval CM. Rodriguez-Morales AJ, et al. Impact of climate variability on the occurrence of leishmaniasis in Northeastern Colombia. Am J Trop Med Hyg. 2006;75:273–277. 11. Randolph SE. Evidence that climate change has caused ‘‘emergence’’ of tick-borne diseases in Europe? Int J Med Microbiol. 2004;293: (Suppl 37): 5–15. 12. Pounds JA, Bustamante MR, Coloma LA, et al. Widespread amphibian extinctions from epidemic disease driven by global warming. Nature. 2006;439:161–167. 13. Taplin D, Lansdell L, Allen AM, et al. Prevalence of streptococcal pyoderma in relation to climate and hygiene. Lancet. 1973; 1:501–503. 14. Yildirim A, Erdem H, Kilic S, et al. Effect of climate on the bacteriology of chronic suppurative otitis media. Ann Otol Rhinol Laryngol. 2005;114:652–655. 15. McBride ME, Duncan WC, Knox JM. Physiological and environmental control of Gram negative bacteria on skin. Br J Dermatol. 1975;93:191–199. 16. Centers for Disease Control and Prevention (CDC). Infectious disease and dermatologic conditions in evacuees and rescue workers after Hurricane Katrina – multiple states, August–September, 2005. MMWR Morb Mortal Wkly Rep. 2005;54:961–964. 17. Rhoads J. Post-Hurricane Katrina challenge: Vibrio vulnificus. J Am Acad Nurse Pract. 2006;18:318–324. 18. Hiransuthikul N, Tantisiriwat W, Lertutsahakul K. Skin and softtissue infections among tsunami survivors in southern Thailand. Clin Infect Dis. 2005;41:e93–96. 19. Garbino J, Garzoni C. Unusual pathogens and multidrugresistant bacteria in tsunami survivors. Clin Infect Dis. 2006;42:889–890. 20. Nieminen T, Vaara M. Burkholderia pseudomallei infections in Finnish tourists injured by the December 2004 tsunami in Thailand. Euro Surveill. 2005;10:E050303.4. 21. Petrini B, Farnebo F, Hedblad MA, et al. Concomitant late soft tissue infections by Cladophialophora bantiana and Mycobacterium abscessus following tsunami injuries. Med Mycol. 2006;44:189–192. 22. Thomas CD, Cameron A, Green RE, et al. Extinction risk from climate change. Nature. 2004;427:145–148.
2 Histology of Microvascular Aging of Human Skin Peter Helmbold
Introduction In this chapter, various histological studies regarding the role of pericytes in the dermis will be summarized, focusing on dermal microvascular aging [1–4]. Aging of the dermis proceeds under special conditions. In addition to chronological aging, a powerful extrinsic factor – chronic UV light – leads to photo-aging (actinic or solar aging). Some known facultative intrinsic or extrinsic factors that influence dermal aging include diabetes mellitus, alcohol, cigarette smoking, and genodermatoses like progeria [5–8]. Previous studies have shown that human dermal microvessel densities depend on age with reduction of functioning reserve capillaries, and there are typical ultrastructural changes in the microvasculature of elderly individuals [5, 6, 9]. Most efforts in microvascular research focus on endothelial cells (EC). By contrast, progress in knowledge on PC which cover microvascular capillaries and venules on their abluminal surfaces has been slow. In the microvasculature, EC and PC are anatomical and functional neighbors. They are separated from each other by the EC basal lamina, which allows punctate direct contact and interdigitation [10]. Endothelin-1 [11] and vascular endothelial growth factors are thought to be the most important cytokines responsible for the interaction of the two cell types [11–13]. Pericytes have contractile function and they are thought to regulate local blood flow [14]. Moreover, they are essential for microvessel stability and control of angioneogenesis [15, 16]. Pericytes are involved in the pathogenesis of diabetic microangiopathy [17, 18], hypertension [19], tumor growth [20], and retinopathy of prematurity [21]. In the skin, PC hyperplasia has been reported in chronic venous insufficiency and in scleroderma [22, 23]. Because of methodological difficulties, most of the dermatological research performed on PC was restricted to ultrastructural or unspecific identification of this cell type by their smooth muscle actin expression [10, 18, 24–26]. One of the most striking methodological problems in this field was identification and counting of a sufficient
number of PC and EC in dermal microvessels. Ultrastructurally, 90–130 ultra-thin sections are required for the reconstruction of one vessel segment with one to four PC [27]. Thus, two methods for identification of cutaneous PC and EC were recently developed: a direct but relatively expensive technique that allows identification of PC and EC nuclei in cryosections by 3G5 antigen and von Willebrand factor expression, and an indirect method that uses identification of PC and EC nuclei according to their anatomical relationship with the collagen IV-positive microvascular basal lamina [2, 22]. The indirect technique in particular allows rapid identification of all key microvascular parameters that were used in this study. From these studies, it can be concluded that the PC/EC ratio is a crucial ‘‘functional-morphological’’ parameter in the dermal microvasculature [22]. In the first study, 120 biopsies from normal skin of 87 patients were obtained from surplus areas (i.e., Burow’s triangle) of routinely excised and histologically controlled benign nevus cell nevi of normal skin (it was previously verified that PC numbers or total microvascular counts were not influenced by non-inflammatory nevus cell nevi, unpublished results). Biopsies with inflammatory cells (infiltrated nevus cell nevi) or histological conditions other than normal skin, and biopsies from patients with known vasculopathies were strictly excluded from the study. To eliminate the influence of latent venous insufficiency, skin samples from the lower legs of patients older than 14 years were generally excluded. Known vascular diseases and diabetes mellitus were additional exclusion criteria. Each specimen was characterized by a set of clinical data: age, sex of the patient, and body location of the biopsy. Further methods are stated in [1]. The relative number of capillaries and venules as well as PC/EC ratios were counted in the upper horizontal dermal plexus including papillar and the upper reticular dermis in collagen IV stained paraffin sections (hematoxylin counterstained) as reported previously [22] (> Fig. 2.1a, b). In short, intra-luminal nuclei (within the lumen that is surrounded by the inner layer of the microvascular basal lamina) were ascribed to EC. By contrast, nuclei found between the two
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layers of microvascular basal lamina were ascribed to PC. Segmented nuclei or nuclei without contact with the basal lamina were excluded. Only clearly recognizable cross or longitudinal sections with unequivocal assignment of the nuclei to the cell types to be determined were selected for examination (> Fig. 2.1a, b). The mean coefficient of variation of this method was 5.7 3.9% [22]. In another experimental study, the ratio PC/EC was studied in clearly cross- or longitudinal-sectioned capillaries by co-localization analysis of 3G5 or vWF binding sites with nuclei in crysoctions by triple-staining with anti-3G5 (a pericyte marker), anti-von Willebrand factor (endothelial marker), and DNA fluorochrome (Hoechst 33258, Sigma) as reported before (> Fig. 2.1c, d) [2, 22]. In a third study part, different TGFs, VEGFs, and PDGFreceptors in the upper dermal plexus were studied in paraffin sections (> Fig. 2.1e, f).
Results and Discussion Two periods of vascular aging – childhood and adulthood. Densities of capillaries and venules in the upper dermal plexus showed dramatic decrease during childhood and slow decrease during adulthood. Results showed a mean of 4.9 2.8 capillaries per HPF and 2.4 1.4 venules per HPF. The density of capillaries was highly negatively dependent on chronological age (r = 0.572, p < 0.001) (> Figs. 2.1a, b and > 2.2). In young children (0–4.99 years) capillary density was 9.7 2.9 per HPF decreasing with adolescence to 4.4 1.5 (15–19.99 years). Thereafter, there was further slow decrease to 2.3 1.4 (range 0.7–5.8) in the age group 70 + years. By contrast, the density of venules showed no significant change during life. At higher ages, the densities of capillaries and venules were comparable. Pericyte loss during childhood and adult chronological life. Mean PC/EC ratios were 0.125 0.054 and 0.132 0.067 in the capillaries and venules, respectively. There was a negative correlation between chronological age and PC/EC ratio of the capillaries (r = 0.560, p < 0.001) or venules (r = 0.594, p < 0.001). Similar to capillary density, the most dramatic changes occurred during adolescence (> Fig. 2.3). In the youngest group (0–4.99 years), PC/EC was twice that in the age group 15–19.99 years, Thereafter, no significant correlation of PC/EC ratio and chronological age was detectable. Studies of the area-based densities of PC and EC showed that the values for PC were highly correlated to the PC/EC ratio, while there was no correlation between total EC counts and PC/EC ratio. It can be concluded that only life-time changes of absolute PC densities (but not changes of EC densities) are responsible for age-dependent
decrease in PC/EC ratio. Fluorescence microscopy analysis of PC and EC distribution by anti-pericyte, anti-endothelial antibodies, and DNA fluorochrome (details see above) brought similar results to those shown in the paraffin imbedded material [1]. Photo-aging. Body regions that reflect typical actinic exposure (photo-aging) showed a negative correlation to each of the key microvascular parameters (capillary density: Spearman r = 0.203, p = 0.039; capillary PC/EC ratio r = 0.242, p = 0.042; venular PC/EC ratio r = 0.255, p = 0.010). The effect of photo-aging was more clearly demonstrable by a newly introduced technique, the histological scoring of dermal basophilic degeneration (DBD, see Chapter 99). The influence of DBD on key microvascular parameters was studied in 84 biopsies of normal skin of subjects 15 years or older. In connection with chronological aging, additional actinic aging could be demonstrated: the capillary density and the PC/EC ratios of the capillaries or venules showed clear diminution with the degree of DBD – a significant photoeffect that is ‘‘added’’ individually to the chronological aging (> Fig. 2.4). Logistic regression demonstrated that PC/EC ratio of the capillaries and venules was predicted by DBD, and, in contrast to younger ages, the chronological age had only weak independent influence in any subjects 15 years or older [1]. Study of TGF-b, PDGFR, and VEGF expressions showed that there was a correlation between microvascular TGF-b1 expression and the PC/EC ratios of capillaries or venules (Spearman r = 0.583, p = 0.006, or r = 0.857, p < 0.001) (> Figs. 2.1e, f and > 2.5), but there was no correlation between the microvascular expression of TGF-b2 and the anatomical parameters of the microvessels. Constitutive microvascular PDGFR-a and -b as well as VEGF expressions were very low and not correlated with any of the microvascular anatomical parameters. In summary, two key parameters of microvasular aging were identified: capillary density and the quantitative ratio of pericytes and endothelial cells (PC/EC ratio). During the first 15 years of life, the number of capillaries of the upper dermis and the PC/EC ratio of the capillaries and venules decrease dramatically by nearly one half. This is called juvenile aging, which might be a process of maturation. Obviously, this maturation is finished with the end of longitudinal body growth. Nevertheless, decrease in capacity of wound healing at the same time advocates classification of this process as an early aging process. This would explain the known deceleration of wound healing and angioneogenesis as well as the reduction of local microvascular reactivity due to aging of the skin by reduction of PC-dependent microvascular
Histology of Microvascular Aging of Human Skin
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. Figure 2.1 Age dependency of microvascular density, pericyte-to-endothelial cell ratio (PC/EC), and microvascular TGF-b1 expression Left column (a, c, e), younger; right column (b, d, f), older skin biopsies. (a) Nuclei of microvascular pericytes (arrows) are identifiable between the two layers of the collagen IV-positive microvascular basal lamina (red) in a collagen IV/hemalaun stained paraffin section of a biopsy of a 21-month-old girl (7.0 capillaries per HPF, PC/EC ratio 0.345). (b) Biopsy from the de´collete´ of a 30-year-old woman with low capillary and pericyte densities (3.1 capillaries per HPF, PC/EC ratio 0.081). (c) Fluorescence microphotograph of pericytic surface 3G5 mAB-binding sites (red), endothelial cell von Willebrand factor (green), and DNA fluorochrome (blue). The figure shows a venular capillary of the upper dermis of a 5-year-old boy with almost complete covering of endothelial cells by pericytes (10.75 capillaries per HPF, PC/EC ratio 0.250). (d) By contrast, a capillary of the upper dermal plexus of a thoracic biopsy of a 31-year-old woman demonstrates sparse PC density (4.25 capillaries per HPF, PC/EC ratio 0.075). (e) High number of microvascular cells express cytoplasmatic TGF-ß1 (arrows) in a PC-rich biopsy of a 7-year-old boy (7.1 capillaries per HPF, PC/EC ratio 0.191). (f) By contrast, a biopsy from the back of a 20-year-old woman with low microvascular and PC densities is lacking microvascular TGF-ß1 (3.5 capillaries per HPF, PC/EC ratio 0.121). The epidermis serves for intrinsic positive control. Scale bar: A, B = 50 mm, C, D = 15 mm, E = 100 mm, F = 200 mm. (Published in Helmbold et al. [1]. Reprinted with permission of J Invest Dermatol)
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. Figure 2.2 Age dependency of the densities of capillaries and venules in the upper dermal plexus age groups: ‘‘0–4’’ means 0–4.99 years etc. (Published in Helmbold et al. [1]. Reprinted with permission of J Invest Dermatol)
. Figure 2.3 Age dependency of the pericyte-to-endothelial cell ratios (PC/EC) of the capillaries and venules of the upper dermal plexus Age groups: ‘‘0–4’’ means 0–4.99 years etc. (Published in Helmbold et al. [1]. Reprinted with permission of J Invest Dermatol)
angioneogenic plasticity and functional loss of physiological microvascular contractility [28–30]. Thereafter, chronological aging alone has a comparatively low influence on both parameters. However, after puberty, the microvascular parameters are modified severely by photo-aging, resulting in further decrease of capillary densities and PC/EC ratios. Regarding the capillary densities, this is consistent with previous
investigations showing higher influence of photo-aging than chronological aging on the upper dermal microvasular plexus during adult life [5, 6, 9]. In summary, two phases of microvascular aging can be postulated in human dermis: a juvenile phase finished by the onset of puberty, when an ‘‘adult plateau’’ is reached, and an adult phase that highly reflects photo-aging with interindividual sunexposure-specific alterations.
Histology of Microvascular Aging of Human Skin
. Figure 2.4 PC/EC ratios of capillaries and venules of the upper dermal plexus in the context of basophilic degeneration, an indicator of photo-aging Age groups: ‘‘0–4’’ means 0–4.99 years, etc. (Published in Helmbold et al. [1]. Reprinted with permission of J Invest Dermatol)
Most important, PC loss, but not changes in EC density, is the cause of the changes in the PC/EC ratio in both the juvenile and the photo-aging of microvessels. PC express several cytokines, particularly TGF-b [31, 32]. The classical members of the TGF-b family belong to a much larger group. In humans, this family consists of almost 30 members, including bone morphogenic proteins, activins, and Mullerian inhibiting substance [33]. These TGF-b family members have effects during development, affect proliferation, differentiation, and cell death, and are important for the development of many tissues. Dermal TGF-b was constitutively active in and around microvessels. A correlation was found between microvascular TGF-b1 expression and the PC/EC ratio. This agrees with previous papers hypothesizing that PCs are the main source of constitutional TGF-b expression within the microvasculature [22,34].
Conclusion Thus, it is concluded that TGF-b1 expression reflects the functional state of the microvessels. TGF-b has different
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. Figure 2.5 Microvascular TGF-b1 expression and EC/PC ratios of the capillaries and venules of the upper dermal plexus White, capillaries; gray, venules. Level 1 and 2 expressions were summarized to ‘‘positive’’ (pos.), and level 0 is represented in the figure as ‘‘negative’’ (neg.). (Published in Helmbold et al. [1]. Reprinted with permission of J Invest Dermatol)
effects on microvessels: it inhibits proliferation and migration of endothelial cells, stimulates in-vivo angiogenesis in the presence of an inflammatory response and increases the stability of blood vessels. Furthermore, it has great impact on fibroblasts and connective tissue through chemotaxis of monocytes and fibroblasts, supporting anchorage-independent growth of fibroblasts, production of antiproteolytic activity via modulation of uPA/PAI-1 expression levels, inhibition of the production of proteases, and stimulation of the production of protease inhibitors [data reviewed [34]]. Thus, results on microvascular TGF-b expression link the relative and absolute absence of microvascular PC in adult and photo-aged skin to proteolytic degradation of dermal connective tissue and reduction of fibroblast function. PC loss might be crucial for dermal connective fiber aging. In contrast to TGFs, significant expression of PDGFR and VEGF seems to be limited to (neo)angiogenesis or cell proliferation, respectively. There was no evidence for the necessity of these cytokines for physiological microvessel maintenance in normal skin.
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Cross-references > Basophilic
(Actinic) Degeneration of the Dermis: An Easy Histological Scoring Approach in Dermal Photoaging
References 1. Helmbold P, Lautenschlager C, Marsch W, et al. Detection of a physiological juvenile phase and the central role of pericytes in human dermal microvascular aging. J Invest Dermatol. 2006;126:1419–1421. 2. Helmbold P, Wohlrab J, Marsch WC, et al. Human dermal pericytes express 3G5 ganglioside – a new approach for microvessel histology in the skin. J Cutan Pathol. 2001;28:206–210. 3. Helmbold P, Fiedler E, Fischer M, et al. Hyperplasia of dermal microvascular pericytes in scleroderma. J Cutan Pathol. 2004;31: 431–440. 4. Helmbold P. Methodische Grundlagen zur Erforschung von Perizyten der Haut. In: Medizinische Fakulta¨t. Halle (Saale): MartinLuther-Universita¨t Halle – Wittenberg. 2002. 5. Braverman IM. Elastic fiber and microvascular abnormalities in aging skin. Clin Geriatr Med. 1989;5:69–90. 6. Korkushko OV, Sarkisov KG. Age-specific characteristics of microcirculation in middle-and old age. Kardiologiia. 1976;16:19–25. 7. Herrick AL, Moore T, Hollis S, et al. The influence of age on nailfold capillary dimensions in childhood. J Rheumatol. 2000;27:797–800. 8. Leung WC, Harvey I. Is skin ageing in the elderly caused by sun exposure or smoking? Br J Dermatol. 2002;147:1187–1191. 9. Chung JH, Yano K, Lee MK, et al. Differential effects of photoaging vs intrinsic aging on the vascularization of human skin. Arch Dermatol. 2002;138:1437–1442. 10. Braverman IM. Ultrastructure and organization of the cutaneous microvasculature in normal and pathologic states. J Invest Dermatol. 1989;93:2S–9S. 11. Dehouck MP, Vigne P, Torpier G, et al. Endothelin-1 as a mediator of endothelial cell-pericyte interactions in bovine brain capillaries. J Cereb Blood Flow Metab. 1997;17:464–469. 12. Takagi H, King GL, Robinson GS, et al. Adenosine mediates hypoxic induction of vascular endothelial growth factor in retinal pericytes and endothelial cells. Invest Ophthalmol Vis Sci. 1996;37:2165–2176. 13. Kim Y, Imdad RY, Stephenson AH, et al. Vascular endothelial growth factor mRNA in pericytes is upregulated by phorbol myristate acetate. Hypertension. 1998;31:511–515. 14. Hirschi KK, D’Amore PA. Pericytes in the microvasculature. Cardiovasc Res. 1996;32:687–698. 15. Lindahl P, Johansson BR, Leveen P, et al. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science. 1997;277: 242–245. 16. Hirschi KK, D’Amore PA. Control of angiogenesis by the pericyte: molecular mechanisms and significance. Exs. 1997;79:419–428. 17. de Oliveira F. Pericytes in diabetic retinopathy. Br J Ophthalmol. 1966;50:134–143.
18. Braverman IM, Sibley J, Keh A. Ultrastructural analysis of the endothelial-pericyte relationship in diabetic cutaneous vessels. J Invest Dermatol. 1990;95:147–153. 19. Wallow IH, Bindley CD, Reboussin DM, et al. Systemic hypertension produces pericyte changes in retinal capillaries. Invest Ophthalmol Vis Sci. 1993;34:420–430. 20. Schlingemann RO, Rietveld FJ, Kwaspen F, et al. Differential expression of markers for endothelial cells, pericytes, and basal lamina in the microvasculature of tumors and granulation tissue. Am J Pathol. 1991;138:1335–1347. 21. Benjamin LE, Hemo I, Keshet E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development. 1998;125:1591–1598. 22. Helmbold P, Fiedler E, Fischer M, et al. Hyperplasia of dermal microvascular pericytes in scleroderma. J Cutan Pathol.2004;31: 431–440. 23. Laaff H, Vandscheidt W, Weiss JM, et al. Immunohistochemical investigation of pericytes in chronic venous insufficiency. Vasa. 1991;20:323–328. 24. Lugassy C, Eyden BP, Christensen L, et al. Angio-tumoral complex in human malignant melanoma characterised by free laminin: ultrastructural and immunohistochemical observations. J Submicrosc Cytol Pathol. 1997;29:19–28. 25. Tsukamoto H, Mishima Y, Hayashibe K, et al. Alpha-smooth muscle actin expression in tumor and stromal cells of benign and malignant human pigment cell tumors. J Invest Dermatol. 1992;98:116–120. 26. Sundberg C, Ivarsson M, Gerdin B, et al. Pericytes as collagenproducing cells in excessive dermal scarring. Lab Invest. 1996;74: 452–466. 27. Braverman IM, Sibley J. Ultrastructural and three-dimensional analysis of the contractile cells of the cutaneous microvasculature. J Invest Dermatol. 1990;95:90–96. 28. Gendron RL. A plasticity for blood vessel remodeling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Surv Ophthalmol. 1999;44:184–185. 29. Scho¨nfelder U, Hofer A, Paul M, et al. In situ observation of living pericytes in rat retinal capillaries. Microvasc Res. 1998;56:22–29. 30. Erber R, Thurnher A, Katsen AD, et al. Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms. FASEB J. 2004;18:338–340, Epub 2003 Dec;2004. 31. Sato Y, Rifkin DB. Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture. J Cell Biol. 1989;109:309–315. 32. Antonelli-Orlidge A, Saunders KB, Smith SR, et al. An activated form of transforming growth factor beta is produced by cocultures of endothelial cells and pericytes. Proc Natl Acad Sci USA. 1989;86:4544–4548. 33. Massague J, Blain SW, Lo RS. TGFbeta signaling in growth control, cancer, and heritable disorders. Cell. 2000;103:295–309. 34. Papetti M, Herman IM. Mechanisms of normal and tumor-derived angiogenesis. Am J Physiol Cell Physiol. 2002;282:947–970.
22 Hyaluronan and the Process of Aging in Skin Robert Stern
Introduction The process of aging of human skin is not well understood. However, loss of apparent moisture is one of the hallmarks of aging skin, with its attendant wrinkling and deterioration in appearance. Hyaluronan (HA, hyaluronic acid) is the predominant mechanism for skin moisture, and must be involved in the aging process. Hyaluronan has an enormous volume of associated water-of-hydration. The water that surrounds the HA molecule is not in equilibrium with the remaining water of the body, but comprises its own compartment. A 70 kg individual has 15 g of HA, half of which is contained in skin. There is also rapid turnover of HA, with a half-life in skin of 1–2 days [1, 2]. However, the biology of skin HA and its bound water has never been thoroughly studied as a function of age. Understanding the metabolism of HA, its reactions within skin, and the interactions of HA with other skin components will facilitate understanding of skin aging as well as decrease in skin hydration. In the past several decades, the constituents of skin have become well characterized. The earliest studies were devoted to the cells that make up skin: epidermis, dermis, and the underlying subcutis. Now it is appreciated that the materials that lie between cells, the matrix components, have major instructive roles for cell and tissue activities. Though the extracellular matrix (ECM) appears amorphous by light microscopy, it forms a highly organized structure of glycosaminoglycans (GAGs), proteoglycans, glycoproteins, peptide growth factors, and structural proteins such as collagen and to a lesser extent, elastin. It is in this ECM that most of skin HA is located. In fact, the predominant component of skin ECM is HA. Recent progress in the details of HA metabolism can also clarify the long appreciated observation that the oxidative damage from free radicals and reactive oxygen species, and the sun damage caused by ultraviolet light cause premature aging of skin. These processes utilize mechanisms similar to that of normal aging, with HA being a common denominator. Attempts to enhance the moisture content of skin, in the most elemental terms, require increasing the level and
the length of time HA is present in skin, preserving optimal chain length of the polymer, and inducing expression of the best profile of HA-binding proteins to decorate the molecule. All of these components are examined in this chapter.
Structure of Hyaluronan Hyaluronan was identified by Karl Meyer [3] in 1938 as a hexuronic acid-containing material that provided the turgor for the vitreous of the eye. It required 20 years, however, before the chemical structure of HA was established. It was later found to be present in virtually every vertebrate tissue. Hyaluronan is a high molecular weight, very anionic polysaccharide. It is a straight chain GAG composed of repeating alternating units of glucuronic acid and N-acetylglucosamine, all connected by b-linkages, GlcAb (1 ! 3) GlcNAc b (1 ! 4), that can reach 107 Da in molecular size. Hyaluronan is the simplest of the GAGs, the only one not covalently linked to a core protein, not synthesized by way of the Golgi pathway, and the only nonsulfated GAG [4, 5]. The b-linkage is of more than passing interest and not merely a curiosity relevant only to carbohydrate chemists. Glycogen is a polymer of a-linked glucose. Changing to a b-linkage converts the polymer to cellulose. A high molecular weight chain of b-linked N-acetylglucosamine is the structure of chitin. Chitin and cellulose are the most abundant sugar polymers on the surface of the earth. Yet such b-linked sugar polymers are not abundant in vertebrate tissues, and the enzymes for their catabolism exist in some suppressed state, for their substrates can survive eons of time. Hyaluronan occurs covalently bound to proteins such as inter-alpha trypsin inhibitor, a plasma protein that also functions as a stabilizer of HA-rich structures, such as the cumulus mass surrounding the mammalian ovum. The molecular domain of HA encompasses a large volume of water that expands extracellular space, hydrates
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_22, # Springer-Verlag Berlin Heidelberg 2010
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tissues, and in the dermis is responsible for skin moisture. It is also a major component in the edema of the inflammatory response. Hyaluronan is capable of expanding its solvent domain over 1,000 times its actual polymer volume. Even at low concentrations, solutions of HA have very high viscosity. By electron microscopy, HA is a linear polymer. It is polydisperse, but usually has a molecular mass of several millions. In solution at physiological pH and salt concentrations, HA is an expanded random coil with an average diameter of 500 nm. Existing models suggest that for high molecular mass HA, the super molecular organization consists of networks in which molecules run parallel for hundreds of nanometers, giving rise to flat sheets and tubular structures that separate and then join again into similar aggregates. There is strong evidence that an H2O bridge between the acetamido and carboxyl groups is involved in the secondary structure. The hydrogen-bonded secondary structure also shows large arrays of contiguous –CH groups, giving a hydrophobic character to parts of the polymer that may be significant in the lateral aggregation or self-association, and for interaction with membranes. This hydrophobic character is perhaps involved in the extrusion of newly synthesized HA chains from the cytoplasmic surface of the plasma membrane where the HAS are located, through the membrane to the exterior of the cell. The unusually stiff tertiary polymeric structure is also stabilized by such hydrophobic interactions.
Functions of Hyaluronan Hyaluronan, despite the simplicity of its structure, has a surprisingly wide range of functions. In high concentrations, as found in the ECM of both the dermis and the epidermis, it regulates water balance and osmotic pressure, functions as an ion exchange resin, and regulates ion flow. It functions as a sieve, to exclude certain molecules, to enhance the extracellular domain of cell surfaces, particularly the luminal surface of endothelial cells. It can function as a lubricant and as a shock absorber. Hyaluronan can also act as a structural molecule, as in the vitreous of the eye, in joint fluid, and in Wharton’s jelly. Hyaluronan promotes cell motility, suppresses cell– cell interactions, and regulates cell–matrix adhesion, promotes proliferation, and suppresses differentiation. It participates in such fundamental processes as embryological development and morphogenesis, wound healing, repair and regeneration, and inflammation. Hyaluronan levels increase in response to severe stress, and in tumor
progression and invasion. Recent studies indicate that HA can also exist intra-cellularly [6]. The intracellular functions of HA are unknown. The persistent presence of HA also inhibits cell differentiation, creating an environment that instead promotes cell proliferation. The elevated levels of anti-adhesive surface HA that promotes cell detachment, also permits the embryonic cell to migrate or the tumor cell to move and metastasize. The water-of-hydration also opens up spaces creating a permissive environment for such cell movements. The ECM that surrounds cells also contains variable levels of HA. It is composed predominantly of structural proteins such as collagen and elastin, as well as proteoglycans, and a number of glycoproteins. The HA content is greatest in embryonic ECM, and in tissues undergoing rapid turnover and repair. The basal lamina or basement membrane that separates dermis and epidermis is also considered an ECM structure. The basal lamina contains HA, though the precise structural position is not known. Loss of basement membrane HA in the skin of diabetic patients correlates with skin stiffness. A number of growth factors are embedded in ECM, concentrated by ECM components where they are protected from degradation. Such factors are presented to cells as mechanisms for growth control and modulators of cell function. Heparan sulfate-containing proteoglycans bind members of the FGF and EGF family, while HA can bind growth factors such as TGF-beta, and also protect them from proteolytic digestion [7]. A complex picture is emerging suggesting that the two classes of GAGs, HA, and heparan sulfate, have opposing functions. An HA-rich environment is required for the maintenance of the undifferentiated, pluripotential state, facilitating motility and proliferation, and abundant in the stem cell niche, while the heparan sulfate proteoglycans promote differentiation. However, the concentration of HA in the ECM can vary widely. Even when the levels are decreased, as in areas of marked fibrosis, HA functions as an organizer of the ECM, as a scaffold about which other macromolecules of the ECM orient themselves. Diameters of collagen fibers can be modulated by levels of HA, the thinner more delicate fibers being favored in regions of high HA concentrations. In fibroblast cultures, the addition of exogenous HA to the medium decreases the diameter of the collagen fibers that accumulate. The ability of HA to promote cell proliferation is dependent in part on the size of the HA molecule, opposite effects being achieved at high and intermediate sizes. High molecular weight HA is anti-angiogenic, while
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intermediate molecular weight HA moieties are highly angiogenic, stimulating growth of endothelial cells, attracting inflammatory cells, and also inducing expression of inflammatory cytokines. Partially degraded HA may have the opposite effect, possibly because it is no longer able to retain and release growth factors such as TGF-b [7]. Such observations are relevant for understanding aspects of skin pathology. For example, the intense staining for HA in psoriatic lesions may in part be due to partially degraded highly angiogenic HA, and may be the mechanism for the marked capillary proliferation and inflammation that characterizes these lesions [8]. Attempts to stimulate HA deposition for purposes of promoting skin hydration and to reverse the effects of aging must use caution that the HA deposited should be of a high molecular weight. This can be done by preventing free radical catalyzed chain breaks and by restricting the catabolic reactions of the hyaluronidases carefully. The most recent development is the realization that HA and associated hyaldherins are intracellular, and have major effects on cellular metabolism. Much of the recent advance comes from the ability to remove the ECM of cultured cells using the highly specific Streptomyces hyaluronidase. Permeabilizing such cells and using confocal microscopy makes it possible to use localization techniques for the identification of intracellular HA and its associated proteins. Some of these intracellular HA complexes appear to be a component of the nuclear matrix in a wide variety of cells. They may have importance in regulating the cell cycle and gene transcription. But no definitive functions have been demonstrated to date. The abilities of HA to associate with itself, with cell surface receptors, with proteins, or with other GAGs speak to the versatility of this remarkable molecule. The tight regulation required for HA deposition in association with these multiple and diverse processes depends on net levels of synthesis and degradation. Hyaluronan is generally produced in the interstitium, in the mesenchymal connective tissue of the body, and is largely a product of fibroblasts. It reaches the blood through the lymphatics. Most of the turnover of HA, approximately 85%, occurs in the lymphatic system. The remaining 15% that reaches the blood stream has a rapid turnover with a t1/2 of 3 to 5 min, being rapidly eliminated by receptors in the liver, and also, by unknown mechanisms in the kidney. When the hepatic or renal arteries are ligated, there is an immediate rise in the level of circulating HA [8]. Thus, humans synthesize and rapidly degrade several grams of HA daily. During acute stress, such as in shock, septicemia, major trauma, and in burn patients there is a rapid rise
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in circulating HA [9]. Such HA may function as a volume expander, as a survival mechanism to prevent circulatory collapse. Some of this rapid rise in HA represents HA recruited from interstitial stores and from lymphatics, and not entirely a reflection of increased synthesis or decreased degradation. However, higher plasma levels of HA do correlate with decreased turnover rates, the t1/2 reaching 20 to 45 min in situations of acute stress. The mean serum and plasma level of HA in healthy young people is 20 to 40 mg/L. This value increases with age and probably reflects slower clearance, and decreased HA degradative capacity, though this has not been carefully investigated. Hyaluronan also increases in the circulation in liver disease, particularly cirrhosis, and in renal failure reflecting aberrant degradation, in rheumatoid arthritis and consistently in some malignancies as a result of increased tumor tissue synthesis.
Hyaluronan Oligomers Size-Specific Activities The extracellular high molecular weight HA polymers are space-filling molecules that hydrate tissues, and are antiangiogenic. These HA polymers are also anti-inflammatory and immunosuppressive. This derives in part from the space-filling polymers’ ability to prevent ligand access to cell surface receptors. The 20 kDa fragments, are highly angiogenic, and stimulate synthesis of inflammatory cytokines. These HA fragments induce transcription of MMPs (matrix metalloproteases), and stimulate endothelial recognition of injury. Oligomers, in the 6 to 20 kDa size range induce inflammatory gene expression in mononuclear and in dendritic cells. Hyaluronan fragments thus are highly angiogenic, inflammatory, and immunostimulatory. Very small HA oligosaccharides also have specific activities. Tetrasaccharides induce expression of heat shock proteins, are antiapoptotic, suppressing cell death. These smallest fragments of HA catabolism thus ameliorate the effects of the intermediate angiogenic and inflammatory fragments. Nature has apparently devised mechanisms to control the extent of stress reactions, to keep them in check. From all of these observations, it can be concluded that fragmentation of HA in the course of its catabolic pathway generates products that are involved in essential processes, with size-specific and widely differing and sometimes opposing biological activities. The working assumption is that HA catabolism is a highly ordered, carefully controlled process, the mechanism for which relies on regulation of the individual enzyme activities. It can be
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concluded that HA fragments are an information-rich system [10].
The Hyaluronan Binding Proteins, the Hyaladherins Hyaluronan exists in a number of states in a vertebrate body. Within the ECM, it can be firmly intercalated within proteoglycans and binding proteins in a bottlebrushlike configuration. It can be bound to cells by means of cell surface receptors. Some of the HA exists in a free form circulating in the lymphatic or cardiovascular system. However, even in this relatively free form, there are a number of binding proteins that decorate HA. These are referred to collectively as hyaladherins [11]. The hyaladherins associate with HA through electrostatic or covalent bonds. It is likely that some of the properties attributed to HA are also a function of the hyaladherins bound to HA. Growth factors, collagens, and many other proteins have been identified. Changes in hyaladherin profiles as a function of aging, and in particular, aging skin, have not been investigated, but are obviously important if the mechanism of skin aging has to be understood.
Hyaluronan Receptors The HA receptors, exist in a myriad of forms, owing their diversity to both variant exon expression as well as to multiple post-translational modifications. The multiple sites for the control of HA synthesis, deposition, celland protein-association, and degradation is a reflection of the complexity of HA metabolism. Their relationships are becoming clarified through the ability to sequence rapidly using the newer techniques of molecular genetics. There promises to be an enormous increase in information and in the understanding of HA biology, as the genes for these enzymes and proteins become known, and rapid sequence analysis carried out.
CD44 There are varieties of HA-binding proteins that are broadly distributed, and with wide variations in locations, in the ECM, cell surface-associated, intracellular, both cytoplasmic and nuclear. The same molecule may occur in multiple locations. However, it is those that attach HA to the cell surface that constitute receptors. The most prominent among these is CD44 [12], a transmembrane glycoprotein
that occurs in a wide variety of isoforms, products of a single gene with variant exon expression. CD44 is coded for by ten constant exons, plus from 0 to 10 or 12 variant exons, depending on species, all inserted into a single extracellular position near the membrane insertion site. Additional variations in CD44 can occur as a result of posttranslational glycosylation, addition of various GAGs, including chondroitin sulfate and heparan sulfate. CD44 is able to bind a variety of other ligands, some of which have not yet been identified. CD44 has been shown, however, to interact with fibronectin, collagen, and heparan-binding growth factors. CD44 is distributed widely, being found on virtually all cells except red blood cells. It plays a role in cell adhesion, migration, lymphocyte activation and homing, and in cancer metastasis. The appearance of HA in dermis and epidermis parallels the histolocalization of CD44. The nature of the CD44 variant exons in skin at each location has not been described. The ability of CD44 to bind HA can vary as a function of differential exon expression. It would be of intrinsic interest to establish what modulation occurs in CD44 variant exon expression with changes in the state of skin hydration, and as a function of age. Only one of the many possible examples of the importance of CD44-HA interactions in normal skin physiology is given here. The HA in the matrix surrounding keratinocytes serves as an adhesion substrate for the Langerhans cells with their CD44-rich surfaces, as they migrate through the epidermis. In skin pathophysiology, the effect of local and systemic immune disorders on such interactions between Langerhans cells and keratinocytes also awaits explication.
RHAMM The other major known receptor for HA is the receptor for HA-mediated motility (RHAMM). This receptor is implicated in cell locomotion, focal adhesion turnover, and contact inhibition. It also is expressed in a number of variant isoforms [13]. The interactions between HA and RHAMM regulate locomotion of cells by a complex network of signal transduction events and interaction with the cytoskeleton of cells. It is also an important regulator of cell growth. The TGF-b stimulation of fibroblast locomotion utilizes RHAMM. TGF-b is a potent stimulator of motility in a wide variety of cells. In fibroblasts, TGF-b triggers the transcription, synthesis, and membrane expression of not only RHAMM, but also the synthesis and expression of
Hyaluronan and the Process of Aging in Skin
the HA, all of which occurs coincident with the initiation of locomotion. In summary, both RHAMM and CD44 may be among the most complex of biological molecules, with locations in an unusually wide variety of cell compartments, and associated with a spectrum of activities involving signal transduction, motility, and cell transformation. The apparent inconsistency of observations between different laboratories regarding the receptors CD44, and RHAMM reflects the subtle ways HA exerts its broad spectrum of biological effects and the myriad of mechanisms for controlling levels of HA expression and deposition. Particularly in the experimental laboratory situation, minor changes in culture conditions, differences in cell passage number, length of time following plating, variations in growth factors contained in lots of serum, or differences in stages of cell confluence have major repercussions in expression of HA, its receptors, or the profile of that decorate the HA molecule. This makes age-related changes in cultured skin cells all the more daunting. One of the major challenges is to identify the profile of hyaladherins specific for the HA of epidermis and dermis, to characterize these proteins and to understand their function in relation to age-related changes. In an examination of skin as a function of age, the levels of HA did not decrease, as would be expected, but rather the binding of HA to tissue proteins became more tenacious, and the HA became increasingly more difficult to extract [14]. Another challenge is to understand how HA as a substrate for degradation by hyaluronidases is affected by associated hyaladherins. It is also reasonable to assume that the secondary structure of the HA polymer is modulated, in part, by the hyaladherins bound to it. A CD44-deficient mouse has been obtained that has a reasonably normal phenotype, suggesting that other HA receptors may substitute for CD44 [15]. In fact, it has been documented that RHAMM is up-regulated when CD44 is deficient. Other receptors including layillin, endothelium receptor (LYVE-1), and others have now been identified using database mining approaches. A convenient tabulation of hyaladherins and HA receptors including database information has recently become available [16].
Hyaluronan and Skin General Observations Hyaluronan occurs in virtually all vertebrate tissues and fluids, but skin is the largest reservoir of body HA, containing more than 50% of the total. Earlier studies on the
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distribution of HA in skin, using histolocalization techniques, seriously underestimated HA levels. Formalin is an aqueous fixative, and much of the soluble tissue HA is eluted by this procedure. The length of time tissue in the formalin is a variable that may explain the conflicting results that are often encountered. Acidification and addition of alcohol to the fixative causes the HA to become more avidly fixed, so that subsequent aqueous steps are unable to elute HA out of the tissue [17]. Comparisons have been made of HA localization in skin sections fixed with acid–formalin/ethanol and conventional formalin fixation. Much of the HA, particularly in the epidermis, is eluted during the process of formalin fixation. This suggests that epidermal HA is more loosely associated with cell and tissue structures than is dermal HA. A further incubation of 24 h in aqueous buffer further increases the disparity between the acid–formalin/alcohol and the conventional fixation technique. Once the tissue has been exposed to the acid–formalin/alcohol, the HA association with tissue becomes tenaciously fixed, with little loss of apparent HA observed following additional aqueous incubation, while the formalin-fixed tissues demonstrate progressive loss of HA.
Epidermal Hyaluronan Until recently, it was assumed that only cells of mesenchymal origin were capable of synthesizing HA, and HA was therefore restricted to the dermal compartment of skin. However, with the advent of the specific techniques for the histolocalization of HA, the biotinylated HA-binding peptide, evidence for HA in the epidermis became apparent. In addition, techniques for separating dermis and epidermis from each other permitted accurate measurement of HA in each compartment, verifying that epidermis does contain HA. Hyaluronan is most prominent in the upper spinous and granular layers of the epidermis, where most of it is extracellular. The basal layer has HA, but it is predominantly intracellular, and is not easily leeched out during aqueous fixation. Presumably, basal keratinocyte HA is involved in cell cycling events, while the secreted HA in the upper outer layers of the epidermis are mechanisms for disassociation and eventual sloughing of cells [14]. Cultures of isolated keratinocytes have facilitated the study of epithelial HA metabolism [18]. Basal keratinocytes synthesize copious quantities of HA. When Ca++ of the culture medium is increased from 0.05 to 1.20 mM, these cells begin to differentiate, HA synthesis levels drop, and there is an onset of hyaluronidase activity.
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This increase in calcium that appears to simulate in culture the natural in situ differentiation of basal keratinocytes parallels the increasing calcium gradient observed in the epidermis. There may be intracellular stores of calcium that are released as keratinocytes mature. Alternatively, the calcium stores may be concentrated by lamellar bodies from the intercellular fluids released during terminal differentiation. The lamellar bodies are thought to be modified lysosomes containing hydrolytic enzymes, and a potential source of the hyaluronidase activity. The lamellar bodies fuse with the plasma membranes of the terminally differentiating keratinocytes, increasing the plasma membrane surface area. Lamellar bodies are also associated with proton pumps that enhance acidity. The lamellar bodies also acidify, and their polar lipids become partially converted to neutral lipids, thereby participating in skin barrier function. Diffusion of aqueous material through the epidermis is blocked by these lipids synthesized by keratinocytes in the stratum granulosum, the boundary corresponding to the level at which HA-staining ends. This constitutes part of the barrier function of skin. The HA-rich area inferior to this layer may obtain water from the moisture-rich dermis. And the water contained therein cannot penetrate beyond the lipid-rich stratum granulosum. The HA-bound water in both the dermis and in the vital area of the epidermis is critical for skin hydration. The stratum granulosum is essential for maintenance of that hydration, not only for the skin, but also for the body in general. Profound dehydration is a serious clinical problem in burn patients with extensive losses of the stratum granulosum.
Dermal Hyaluronan The HA content of the dermis is far greater than that of the epidermis, and accounts for most of the 50% of total body HA present in skin. The papillary dermis has the more prominent levels of HA than does reticular dermis. The HA of the dermis is in continuity with both the lymphatic and vascular systems, which epidermal HA is not. Exogenous HA is cleared from the dermis and rapidly degraded. The dermal fibroblast provides the synthetic machinery for dermal HA, and should be the target for pharmacological attempts to enhance skin hydration, and age-related changes. The fibroblasts of the body, the most banal of cells from a histological perspective, are probably the most diverse of all vertebrate cells with the broadest repertoire of biochemical reactions and potential pathways for differentiation. Much of this diversity is site specific. What makes
the papillary dermal fibroblast different from other fibroblasts is not known. However, these cells have an HA synthetic capacity similar to that of the fibroblasts that line joint synovium, responsible for the HA-rich synovial fluid.
Changes in Skin Hyaluronan with Aging The HA levels are high in a fetal circulation and fall shortly after birth. After maintaining a steady level for several decades, circulating levels of HA then begin to increase again in old age. Elevated levels of circulating HA are also found in the syndromes of premature aging, in progeria, and in Werner’s Syndrome. This increase in HA with age is counter-intuitive, and not understood. Increased HA levels in the bloodstream decrease immune competence. Various mechanisms have been invoked. An HA coating around circulating lymphocytes may prevent ligand access to lymphocyte surface receptors. The increased HA may represent one of the mechanisms for the immunosuppression in the fetus. The reappearance of high levels of HA in old age may, similarly, be one of the mechanisms of the deterioration of the immune system in the elderly. The increasing levels of HA with aging may be a reflection of the deterioration of hydrolytic reactions, including the hyaluronidases that maintain the steady state of HA. This is a far more likely mechanism than an increase in HA synthetic activity. Though dermal HA is responsible for most skin HA, epidermal cells are also able to synthesize HA. The most dramatic histochemical change observed in senescent skin is the marked decrease in epidermal HA. In senile skin, HA is still present in the dermis, while the HA of the epidermis has disappeared entirely. The proportion of total GAG synthesis devoted to HA is greater in epidermis than in dermis, and the reasons for the precipitous fall with aging is unknown. The synthesis of epidermal HA is influenced both by the underlying dermis, as well as by topical treatments, such as with retinoic acids, indicating that epidermal HA is under separate controls from dermal HA. In contrast with previous in vitro and in vivo observations, recent studies document that the total level of HA remains constant in skin with aging [14]. The major agerelated change is the increasing avidity of HA with tissue structures with the concomitant loss of HA extractability. Such intercalated HA may have diminished ability to take on water of hydration. This decreased volume of water of hydration HA is obviously a loss in skin moisture. An important study for the future would be to define precisely the hyaldherins, the HA-binding proteins, that decorate
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the HA in senile skin, and to compare that profile with that of young skin, in both the dermal and epidermal compartments. Progressive loss in the size of the HA polymer in skin as a function of age has also been reported. The increased binding of HA with tissue as a function of age parallels the progressive crosslinking of collagen and the steady loss of collagen extractability with age. Each of these phenomena contributes to the apparent dehydration, atrophy, and loss of elasticity that characterizes aged skin.
Photo-Aging Repeated exposure to UV radiation from the sun causes premature aging of skin. UV damage causes initially a mild form of wound healing, and is associated first with elevated dermal HA. As little as 5 min of UV exposure in nude mice causes enhanced deposition, indicating that UV-induced skin damage is an extremely rapid event. The initial ‘‘glow’’ after sun exposure may be a mild edematous reaction induced by the enhanced HA deposition. But the transient sense of well being in the long run extracts a high price, particularly with prolonged exposure. Repeated exposures ultimately simulate a typical wound healing response with deposition of scar-like type I collagen, rather than the usual types I and III collagen mixture that gives skin resilience and pliability. The biochemical changes that distinguish photo-aging and chronological aging have not been identified. The abnormal GAGs of photo aging are those also found in scars, in association with the changes found late in the wound healing response, with diminished HA and increased levels of chondroitin sulfate proteoglycans. There is also an abnormal pattern of distribution. The GAGs appear to be deposited on the elastotic material that comprises ‘‘elastosis’’ and diffusely associated with the actinic damaged collagen fibers. These appear as ‘‘smudges’’ on H&E sections of sun-damaged skin, rather than between the collagen and elastin fibers as would be observed in normal skin.
Oxidative Stress Reactive oxygen species or free radicals are a necessary component of the oxygen combustion that drives the metabolism of living things. Though they are important for generating the life force, they simultaneously are extraordinarily harmful. Organisms had to evolve protective mechanisms against oxidative stress. Over the course of
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evolution, different enzymatic and non-enzymatic antioxidative mechanisms were developed, such as various vitamins, ubiquinone, glutathione, and circulating proteins, for example, hemopexin. Hyaluronan may also be one such mechanism, acting also as a free radical scavenger. Sunlight (UV light) is an additional generator of harmful oxygen-derived species such as hydroxyl radicals. Such radicals have the ability to oxidize and damage other molecules such as DNA causing cross-linking and chain scission. These hydroxyl radicals may also be destructive for proteins and lipid structures, as well as ECM components such as HA. After a very few minutes of UV exposure, disturbance in HA deposition can be detected. An anomalous situation exists, therefore, that HA can both be protective as a free radical scavenger, and at the same time a target of free radical stress. This paradox may be understood by a hypothetical model in which HA protects the organism from the free radical stress generated by the oxygen-generated internal combustion, but is itself harmed by the more toxic free radicals generated by the external world, by UV irradiation. The generation of HA fragments by UV may underlie some of the irritation and inflammation that often accompanies long term or intense sun exposure. As discussed above, HA fragments are themselves highly angiogenic and inflammatory, inducing the production of a cascade of inflammatory cytokines. Further complications have occurred in this assembly of metabolic attack and counter-attack reactions that have been compiled in the selective forces of evolution. Unusually high levels of antioxidants are present in skin, such as Vitamins C and E, as well as ubiquinone and glutathione. However, these precious compounds are depleted by exposure to sunlight. To prevent this sun-induced cascade of oxidative injuries, topical preparations containing antioxidants have been developed in the past several decades. Initially, such antioxidants were added as stabilizers to various dermatologic and cosmetic preparations. In particular, lipophilic Vitamin E has been the favorite as a stabilizing agent. However, following oxidation, Vitamin E is degraded into particularly harmful pro-oxidative metabolites. In the past several years, increasing concentrations of antioxidants have been used in such skin preparations, in an attempt to create complementary combinations, or to create constant recycling pairs that alternatingly oxidize and reduce each other. Finally, molecules such as HA should be protected by topical antioxidants, to prevent degradation. Topical antioxidants, protecting against free radical damage as well as maintaining HA integrity may have major effects against natural aging and photo-aging.
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Inflammation Chronic inflammation also causes premature aging of the skin, as observed in patients with atopic dermatitis. The constant inflammatory process leads to decreased function of the skin barrier, accompanied by loss of skin moisture. Presumably, the skin of such patients contains decreased levels of HA. Alternatively, the HA may reflect that found in chronological aging, with a change in the ability to take on water of hydration with enhanced association with tissue structures and loss of extractability. Demonstration of such changes and the precise histolocalization of this decreased HA deposition would be of intrinsic interest, a study that has not yet been performed. The acute inflammatory process is associated initially with increased HA levels, the result of the cytokines released by the polymorphonuclear leukocytes, the predominant cells of the acute inflammatory process. The erythema, swelling, and warmth of the acute process are followed later by the characteristic dry appearance and the formation of wrinkles. The precise mechanisms are unknown, but may relate to the differences between acute and chronic inflammatory cells and the attendant chemical mediators released by such cells. Alternatively, initiation of a wound healing response, with collagen deposition, may be a mechanism invoked for the premature aged appearance of the skin in chronic inflammation.
Hyaluronan Synthases A single enzyme is now recognized as being able to synthesize HA, dual-headed transferases that utilize alternately the two UDP-sugar substrates, UDP-glucuronic acid, and UDP-N-acetylglucosamine [19]. The HA cytoplasmic product is extruded through the plasma membrane into the extracellular space by means of an ABC transporter system that permits unconstrained polymer growth. Such growth could not occur in the Golgi or on the endoplasmic reticulum where most sugar polymers are synthesized, without destruction of the cell. There are three synthase genes in the mammalian genome, coding for HAS-1, -2, and -3. They are differentially regulated, with each producing a different size polymer. Sequence data of the HAS isoforms suggest that they contain seven membrane-associated regions and a central cytoplasmic domain possessing several consensus sequences that are substrates for phosphorylation by protein kinases. The ABC transporter system proteins required for HA transport through the plasma membrane are encoded at a
chromosomal region immediately adjacent to the HA synthase genes. In situ expression of the HAS-1 and -2 genes are upregulated in skin by TGF-b, in both dermis and epidermis, but there are major differences in the kinetics of the TGF-b response between HAS-1 and -2, and between the two compartments, suggesting that the two genes are independently regulated. This also suggests that HA has a different function in dermis and epidermis. Stimulation of HA synthesis also occurs following phorbol ester (PMA) and PDGF treatment, although a direct effect on HAS has not been demonstrated. Glucocorticoids induce a nearly total inhibition of HAS mRNA in dermal fibroblasts and osteoblasts. Extracts of dermal fibroblasts indicate that HAS-2 is the predominant HA synthase therein. This may be the molecular basis of the decreased HA in glucocortcoid-treated skin. However, an additional effect on rates of HA degradation has not been examined.
Hyaluronidases Hyaluronan is very metabolically active, with a half-life of 3 to 5 min in the circulation, less than 1 day in skin, and even in an inert a tissue as cartilage, the HA turns over with a half-life of 1 to 3 weeks. This catabolic activity is primarily the result of hyaluronidases (HYALs), endoglycolytic enzymes with a specificity in most cases for the b 1–4 glycosidic bond. The hyaluronidase family of enzymes has, until recently, been neglected, in part because of the great difficulty in measuring their activity. They are difficult to purify and characterize, are present at exceedingly low concentrations, have very high specific activities that are unstable in the absence of detergents and protease inhibitors during the purification procedures. Once purified, these enzymes appear to be perfectly stable. New assay procedures have now facilitated their isolation and characterization. The human genome project has also promoted explication at the genetic level, and a virtual explosion of information has ensued [20]. Six hyaluronidase-like sequences are present in the mammalian genome, resulting probably from two duplication events, resulting in three genes, followed by en masse block duplication, generating six hyaluronidase genes. All are transcriptionally active with unique tissue distributions. In the human, three genes (HYAL1, HYAL2, and HYAL3) are found tightly clustered on chromosome
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3p21.3, coding for HYAL1, HYAL2, and HYAL3. Another three genes (HYAL4, PHYAL1 (a pseudogene), and sperm adhesion molecule1 (SPAM1)) are clustered similarly on chromosome 7q31.3. They code respectively for HYAL4, a pseudogene transcribed but not translated in a human, and PH-20, the sperm enzyme. The enzymes HYAL1 and HYAL2 constitute the major hyaluronidases for HA degradation in somatic tissues. HYAL1, an acid-active lysosomal enzyme, was the first somatic hyaluronidase to be isolated and characterized. It is a 57 kDa single polypeptide glycoprotein that also occurs in a processed 45-kDa form, the result of two endoprotease reactions. The resulting two chains are bound by disulfide bonds. This is not a zymogen-active enzyme relationship, since the two isoforms have similar specific activities. Only the larger form is present in the circulation, while both isoforms occur in urine, in tissue extracts, and in cultured cells. Why an acid-active hyaluronidase should occur in plasma is not clear. Some species do not have detectable enzymatic activity in their circulation, but an inactive 70 kDa precursor form of the enzyme is present in such sera, detectable by Western blot. HYAL1 is able to utilize HA of any size as substrate, and generates predominantly tetrasaccharides. HYAL2 may also be acid-active, anchored to plasma membranes by a GPI (glycosyl-phosphatidylinositol)link. HYAL2 occurs also in a processed soluble form. Again, the difference in function between the two isoforms is not known. HYAL2 cleaves high molecular weight HA to a limit product of approximately 20 kDa, or about 50 disaccharide units, while HYAL1 is able to digest the high molecular weight polymer to a limit digestion product consisting predominantly of tetrasaccharides. HYAL1 and HYAL2 have similar structures, and the difference in their reaction products requires explanation. The biological properties of HA in aqueous solution is controlled by reversible tertiary structures, as defined by NMR spectroscopy. Evidence suggests a b-pleated sheetlike array stabilized by H- and hydrophobic bonds. Easy transitions between secondary and tertiary structures occur that are convenient mechanisms for switching between functions. The 20 kDa or 50-disaccharide unit is around the size at which such stable tertiary structures are expected to form. Polymers greater than 20 kDa provide the preferred substrate for HYAL2. The enzyme cleaves at a much slower rate once the HA substrate loses tertiary structure. The hyaladherins may also provide additional substrate specificity. The array of hyaladherins that bind
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to tertiary HA structures may differ from those that bind to HA chains with exclusively secondary structure. The specificity of HYAL2 may depend on a combination of differences in bound hyaladherins and on secondary versus tertiary structure.
Hyaluronidase Inhibitors Macromolecular Inhibitors The extraordinarily rapid turnover of HA in tissues suggests that tightly controlled modes exist for modulating steady state levels of HA. The HA of the vertebrate body is of unique importance, and rapid increases are required in situations of extreme stress. Rapid turnover of HA in the normal state indicates constant synthesis and degradation. Inhibition of degradation would provide a far swifter response to the sudden demand for increased HA levels than increasing the rate of HA synthesis. The ability to provide immediate high HA levels is a survival mechanism for the organism. This may explain the rapid rates of HA turnover that occur in the vertebrate animal under basal conditions. It can be compared to the need to suddenly drive an automobile much faster in the case of an emergency, not by stepping on the accelerator, but by taking a foot off the break. If inhibition of HA degradation by hyaluronidase occurs, then a class of molecules that have not been explored, the hyaluronidase inhibitors, are very important. It can be postulated that with extreme stress, hyaluronidase inhibitors would be found in the circulation as acute phase proteins, the stress response products synthesized by the liver. These would prevent the everpresent rapid destruction and allow levels of HA to quickly increase. Circulating hyaluronidase inhibitor activity has been identified in human serum over half a century ago. Modifications in levels of inhibitor activity have been observed in the serum of patients with cancer, liver disease, and with certain dermatological disorders. This area of biology is unexplored, and though some early attempts were made, these hyaluronidase inhibitors have never been thoroughly characterized at a molecular level. Cultured cells secrete hyaluronidases into the culture media, away from the cells. Such a phenomenon does not occur within tissues. The production of unopposed hyaluronidase activity would cause great havoc in tissues. Simultaneous deposition of hyaluronidases and their inhibitors is a reasonable scenario, one that parallels
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control of the matrix metalloproteinases by their TIMPs (tissue inhibitors of MMPs). An important corollary of such observations would be to examine the nature and changes of hyaluronidase inhibitors that occur as a result of aging.
Low Molecular Weight Inhibitors Classes of lower molecular weight inhibitors of hyaluronidase have been identified, some of which come from folk medicines, from the growing field of ethnopharmacology. Some anti-inflammatories as well as some of the ancient beauty aids and practices for freshening of the skin may have some of these compounds as the basis of their mechanism of action. Clinically, heparin used as an anticoagulant, has potent anti-hyaluronidase activity, as does indomethacin, a classic nonsteroidal anti-inflammatory agent, and salicylates. More recently, dextran sulfate and Vitamin C and some of its derivatives, have been shown to be potent inhibitors of vertebrate hyaluronidases.
Non-Enzymatic Degradation The HA polymer can be degraded nonenzymatically by a free radical mechanism, particularly in the presence of reducing agents such as thiols, ascorbic acid, ferrous, or cuprous ions. This mechanism of depolymerization requires the participation of molecular oxygen. The use of chelating agents in pharmaceutical preparations to retard free radical catalyzed scission of HA chains has validity. However, a carefully monitored effect of such agents on HA chain length in human epidermis has not been attempted. Whether such agents can also affect the integrity of dermal HA in protecting them from free radical damage, and whether these agents have any substantial effect on the moisturizing properties of skin HA remain important questions to be answered. These have major implications for the mechanisms of skin aging.
A Scheme for Hyaluronan Metabolism It is well established that HA is taken up by cells for degradation through the CD44 receptor. The high molecular weight extracellular polymer is tethered to the cell surface by the combined efforts of CD44 and the GPIanchored enzyme HYAL2. The HA-CD44-HYAL2
complex is enriched in specialized microdomains. These are invaginations of the plasma membranes termed lipid rafts, significant because they also recruit a large number of key signaling molecules. One category of lipid rafts is caveolae, structures rich in the proteins caveolin and flotillin. HYAL2 interacts with CD44 and with a Na+-H+ exchanger termed NHE1 that creates an acidic microenvironment for the acidactive hyaluronidase enzyme [21]. The HA is cleaved to the 20 kDa limit products corresponding to about 50 disaccharide units. The CD44, a multifunctional transmembrane glycoprotein that is the predominant HA receptor, is expressed in a number of different isoforms. The variant exons of CD44 specifically involved in the interaction with HYAL2 and NHE1 in the process of HA binding, uptake, and degradation have not been determined. The HYAL2-generated HA fragments are internalized, delivered to endosomes, and ultimately to lysosomes, where HYAL1 degrades the 20 kDa fragments to small disaccharides. Two lysosomal b-exoglycosidases, b-glucuronidase and b-N -acetylglucosaminidase, participate in this degradation.
The Hyaluronasome, a New Mini Organelle Based on the observations described above, it is possible to invoke a new mini-organelle specific for HA metabolism, termed the hyaluronasome. Parallels between glycogen and HA metabolism are the basis of this formulation. A glycogen mini-organelle occurs in both liver and muscle tissues. The hyaluronasome may resemble the glycogen granule, each involved in the metabolism of large carbohydrate structures, glycogen being a branched chain polymer of a-linked sugars, and HA, a straight chain polymer of b-linked sugars. Readily visualized by the electron microscope, glycogen granules appear as bead-like structures localized to specific subcellular locales. Each glycogen granule is a functional unit, not only containing carbohydrate, but also enzymes and other proteins needed for its metabolism. These proteins are not static, but rather associate and dissociate depending on the carbohydrate balance in the tissue. Regulation takes place not only by allosteric regulation of enzymes, but also due to other factors, such as subcellular location, granule size, and association with various related proteins. Such observations may be applicable to the hyaluronasome. A multiprotein membrane-associated
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complex that contains HA synthetic activity has been described. This hyaluronate synthase complex may be a component of the hyaluronasome, containing synthetic as well as catabolic activities, a functional unit that could provide response mechanisms dependent on the metabolic state of the cell. Suggestive evidence comes from several sources. Cultured cells treated with low concentrations of hyaluronidase increase their levels of HA synthesis. Treatment of isolated membrane preparations with low concentrations of hyaluronidase has a similar effect. This is compatible with a feedback mechanism enabling cells to sense levels of HA that have been synthesized. Exogenously added hyaluronidase cleaves newly synthesized HA chains as they are being extruded through the plasma membrane, informing the cell that inadequate amounts of HA have been synthesized. The hyaluronasome, lying just under and partially embedded within the plasma membrane, could rely on a servomechanism using a receptor such as CD44 for relaying such feedback messages. Hyaluronidase treatment of culture cells modulates the profile of expression of CD44 variant exons, thus providing the exquisite controls necessary for such regulatory mechanisms. Levels of HA that cells deposit must respond to various physiological states including growth phase, confluence, inversely related to cell density in both fibroblasts, and keratinocytes, mitosis and cell detachment from the substratum, calcium concentrations, anoxia and lactate, viral transformation, and serum stimulation. Immunolocalization data indicate that some of the HAS enzymes and hyaluronidases colocalize. All of this evidence supports, the existence of the hyaluronasome minirganelle. The hyaluronasome, because of its ability to respond to extracellular events as well as to the intracellular metabolic state of the cell may contain HA receptors, RHAMM and CD44, HA synthase enzymes, the hyaluronidases, and hyaluronidase inhibitors, and HA-binding proteins. The hyaluronasome can regulate levels of HA deposition with great precision by allosteric regulation of the enzymes contained therein utilizing not only hyaladherins and related proteins, but perhaps by posttranslational modifications such as phosphorylation and sulfation. Levels of specific phosphorylated proteins are utilized in the analysis of signaling transduction pathways. However it was the phosphorylases that degrade glycogen and related proteins of glycogen catabolism that provided the paradigm for protein phosphorylation as a control mechanism. Similar modifications applied to the control of HA catabolism would be in that tradition.
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Enhancing Hyaluronan Deposition in Order to Counteract the Aging Process The Lactate Effect Markedly enhanced levels of HA occur in the stroma surrounding malignant tumors. The HA stimulates cell motility and hydrates tissues, creating spaces into which tumor cells can move in the process of invasion and metastatic spread. Lactate is usually the product of anaerobic metabolism. However, cancer cells produce lactate even when oxygen is abundant. The ability of malignant cells to generate lactate, even in the presence of sufficient quantities of oxygen is known as the Warburg effect. Application of lactate to skin is already a pharmacological technique thought to enhance HA deposition. Such preparations are available at both low and high doses, the later requiring a prescription.
Alpha-hydroxy Acids Fruit compresses have been applied to the face as beauty aids for millennia. The alpha-hydroxy acids contained in fruit extracts, tartaric acid in grapes, citric acid in citrus fruits, malic acid in apples, mandelic acid in almond blossoms and apricots are active principles for skin rejuvenation. Such alpha-hydroxy acids stimulate HA production in cultured dermal fibroblasts. The results of such alkaline preparations may depend more on their peeling effects, rather than on the ability of alpha-hydroxy acids to stimulate HA deposition. Lactic acid, citric acid, and glycolic acid, in particular, though frequent ingredients in alpha-hydroxy acidcontaining cosmetic preparations, have widely varying HA-stimulating activity in dermal fibroblasts. Some of these preparations may owe their effectiveness to their traumatic peeling, astringent properties, with constant wounding of the skin. The cosmetic effects of these preparations of alpha-hydroxy acids, including lactic acid, involve increased skin smoothness with the disappearance of lines and fine wrinkles, all of which counteract the aging process. Long-term use, however, results in thickening of the skin, in both the epidermal and papillary dermal layers, because of a mild fibrous reaction. This results from a reaction similar to diffuse wound healing, and explains the increased thickness and firmness of both dermis and epidermis. The increased collagen deposition documented in skin after prolonged use is consistent with a
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wound healing effect. The normal aging process in skin, particularly the coarseness and thickening may resemble some of these reactions.
Vitamin C The structure of ascorbic acid resembles an alpha-hydroxy acid, which is generally not appreciated. Ascorbic acid is present in most fruits, and may underlie some of the effects attributed to fruit extracts. Vitamin C has pronounced HA-stimulating effects in the fibroblast assay. But its antioxidant activity confounds the effects it may induce. The deposition of HA is stimulated when Vitamin C is added to cultured fibroblasts. The most profound changes occur in the compartmentalization of HA. The preponderance of the enhanced HA becomes cell-layer instead of being secreted into the medium. The chemical reactions catalyzed by ascorbic acid that bind HA to cell or matrix components are not known. As aforementioned, derivatives of Vitamin C and its derivatives and analogs can function as hyaluronidase inhibitors. Thus, some of the ability of Vitamin C to enhance HA deposition may be attributed to its inhibition of hyaluronidase activity. Skin preparations containing high concentrations of Vitamin C, as currently in use, may have some validity as anti-aging therapies.
Vitamin A Hyaluronan hinders the onset of differentiation, as discussed earlier. Retinoic acid retards the differentiation of epidermal keratinocytes, as shown in skin organ cultures, a result of the ability of retinoic acid to stimulate HA deposition. Retinoic acid leads to the accumulation of HA in the superficial layers of the epidermis by stimulating HA synthesis specifically in keratinocytes. Some of this accumulation occurs as expanded intercellular HA, which may account for the weakened cohesion of keratinocytes observed both in vivo and in vitro. Topical applications of retinoic acid derivatives reduce the visible signs of aging and of photodamage, though there is little correlation between the histologic changes and the clinical appearance of the skin. Initial improvement in fine wrinkling and skin texture correlates with the deposition of HA in the epidermis. While Vitamin D is considered the ‘‘sunshine vitamin,’’ Vitamin A has been accepted as an apparent antidote for the adverse effects of sun exposure, and assumed to prevent and repair cutaneous photodamage. Application of
Vitamin A derivatives does reverse some of the sun damage to skin, particularly the roughness, wrinkling, and irregular pigmentation. For the over-40 generation, brought up in an era of ‘‘suntan chic,’’ appropriate preparations to restore or to prevent further deterioration of skin are critically important. Impairment of the retinoid signal transduction pathways occurs as a result of prolonged UV exposure. Down regulation of nuclear receptors for Vitamin A occurs, resulting in a functional deficiency of Vitamin A. Application of Vitamin A derivatives would appear to be an obvious treatment modality. Topical application of Vitamin A does increase the HA in the epidermal layer, increasing the thickness of the HA meshwork after prolonged treatment. Vitamin A thus enhances repair, as can be demonstrated in photo-aged hairless mouse model. The decline in GAG, and in particular HA deposition that occurs with UVB irradiation, can be prevented by retinoic acid treatment.
Vitamin E Radical scavengers such as a-tocopherols prevent oxidative degradation of HA. In tissue culture systems, the addition of Vitamin E to the medium prevents spontaneous degradation of HA, as does superoxide dismutase. In Vitamin-E-deficient animals, there is a decrease in GAGs in tissues, including HA. This could be reversed by the addition of Vitamin E to diets, suggesting that tocopherol supplements can enhance HA in human skin, and counteract the aging process.
Vitamin D Vitamin D, and in particular, the hormonally active di-hydroxy form, is a regulator of the proliferation and differentiation of skin cells, including not only epidermal kertainocytes, but also dermal fibroblasts and adipocytes. A result of prolonged UV exposure is dermal fibrosis, the excessive deposition of collagen and other ECM components within the dermis. The commandeering of mesenchymal cells to become fibroblasts, and the conversion of adipocytes to fibroblasts are thought to be the underlying mechanism. Pretreatment of skin with Vitamin D prevents the disappearance of adipocytes and the accumulation of fibroblasts. The appearance of HA, the first step in the wound healing response that initiates the cascade that leads to accumulation of the fibrous reaction, can be prevented by such treatment.
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Steroids Topical and systemic treatment with glucocorticoids induces atrophy of skin causing premature aging, with a concomitant decrease in HA. In human skin organ cultures, hydrocortisone has a bimodal effect. At low physiological concentrations, 109 M, hydrocortisone maintains active synthesis and turnover of HA in the epidermis, while at high concentrations, 105 M, hydrocortisone reduces epidermal HA content. The effect is achieved through both decreased synthesis as well as decreased rates of degradation. The high concentrations of cortisone also enhance terminal differentiation of keratinocytes and reduce rates of cell proliferation. Hydrocortisone is also a potent inhibitor of HA synthesis in fibroblasts. HAS-2 is the predominant synthase of dermal fibroblasts, of the three HA synthase genes. Glucocorticoids induce a rapid and near total suppression of HAS-2 mRNA levels. The inhibition of HA deposition thus appears to occur at the transcriptional level. Progesterone inhibits HA synthesis in fibroblasts cultured from the human uterine cervix. Thus, the steroid effect on HA appears to be system-wide. Hydrocortisone, as well as dexamethasone suppresses the ability of TGF-beta to stimulate HA synthesis through the p38 MAP kinase induced activation of the HAS genes. Edema is one of the four cardinal signs of acute inflammation. The ability of glucocorticoids to suppress inflammation occurs in part by their ability to suppress the deposition of HA, the primary mechanism of edematous swelling that occurs during the inflammatory response. Skin is also an important target organ for estrogens. The estrogenic effect on skin is well characterized, as well as the effect of estrogen withdrawal. A major effect of estrogen is the increased levels of HA deposition and the associated water of hydration. Topical estrogens are also able to enhance HA deposition in skin, as documented in the hairless mouse skin model. The isoflavones found in soy bean extracts, such as genistein and daidzein, that are phytoestrogens, are also able to enhance HA deposition. Their estrogen-like structures may account for their ability to enhance HA deposition. Withdrawal of estrogen production, which occurs following menopause, explains some of the age-related changes in women. Women with increased levels of body fat have skin that ages more slowly following menopause. This may occur because the fat depots function as slow release capsules of estrogens. Such steroids are fat soluble, and are stored during periods of active estrogen production.
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Ethnopharmacology Agents Used to Retard Skin Aging Ethno-pharmaceuticals have long provided Western medicine with a wide variety of drugs that enhance the appearance of skin, and retard the aging process. Many of these function though effect HA metabolism. Fruit compresses have been applied to the skin for thousands of years as a traditional beauty aid. The alpha-hydroxy acids contained in fruit extracts enhance HA deposition through hyaluronidase inhibition. Other examples are the ginsenosides, major active ingredients of ginseng, which when applied topically, induce expression of the HAS-2 gene and increase skin content of HA. A myriad of other such anti-aging agents applied to skin from folk medicines await identification.
Conclusion and Future Perspectives The biology of HA and its metabolic cycles are in their infancy. The enzymatic steps that constitute extracellular and intracellular HA cycles are beginning to be sorted out. The goals that lie ahead are to identify all the reactions involved, and to devise mechanisms for modulating these reactions, with the ultimate goal of enhancing skin appearance and increasing the moisture content of damaged and aging skin.
References 1. Laurent UB, Dahl LB, Reed RK. Catabolism of hyaluronan in rabbit skin takes place locally, in lymph nodes and liver. Exp Physiol. 1991;76:695–703. 2. Reed RK, Lilja K, Laurent TC. Hyaluronan in the rat with special reference to the skin. Acta Physiol Scand. 1988;134:405–411. 3. Meyer K, Palmer JW. The polysaccharide of the vitreous humor. J Biol Chem. 1934;107:629–634. 4. Lee JY, Spicer AP. Hyaluronan: a multifunctional, megaDalton, stealth molecule. Curr Opin Cell Biol. 2000;12:581–586. 5. Toole BP. Hyaluronan is not just ‘‘goo.’’ J Clin Invest. 2000;106: 335–336. 6. Evanko SP, Wight TN. Intracellular localization of hyaluronan in proliferating cells. J Histochem Cytochem. 1999;47:1331–1342. 7. Locci P, Marinucci L, Lilli C, Martinese D, Becchetti E. Transforming growth factor beta 1 hyaluronic acid interaction. Cell Tissue Res. 1995;281:317–324. 8. Kumar S, West DC. Psoriasis, angiogenesis and hyaluronic acid. Lab Invest. 1990;62:664–665. 9. Engstroem-Laurent A, Hellstroem S. The role of liver and kidneys in the removal of circulating hyaluronan. An experimental study in the rat, Connect. Tissue Res. 1990;24:219–224.
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10. Stern R, Asari AR, Sugahara KN. Size- specific fragments of hyaluronan: an information rich system. Eur J Cell Biol. 2006;85:699–715. 11. Day AJ, Prestwich GD. Hyaluronan-binding proteins: tying up the giant. J Biol Chem. 2002;277:4585–4588. 12. Lesley J, Hascall VC, Tammi M, Hyman R. Hyaluronan binding by cell surface CD44. J Biol Chem. 2000;275:26967–26975. 13. Cheung WF, Cruz TF, Turley EA. Receptor for hyaluronan-mediated motility (RHAMM), a hyaladherin that regulates cell responses to growth factors. Biochem Soc Trans. 1999;27:135–142. 14. Meyer LJM, Stern R. Age-dependent changes of hyaluronan in human skin. J Invest Dermatol. 1994;102:385–389. 15. Nedvetzki S, Gonen E, Assayag N, Reich R, Williams RO, Thurmond RL, Huang JF, Neudecker BA, Wang FS, Turley EA, Naor D. RHAMM, a receptor for hyaluronan mediated motility, compensates for CD44 in inflamed CD44-knockout mice: a different interpretation of redundancy. Proc Natl Acad Sci USA. 2004;101:18081–18086.
16. Spicer AP, Tien JY. Hyaluronan and morphogenesis. Birth Defects Res Part C Embryo Today. 2004;72:89–108. 17. Lin W, Shuster S, Maibach HI, Stern R. Patterns of hyaluronan staining are modified by fixation techniques. J Histochem Cytochem. 1997;45:1157–1163. 18. Passi A, Sadeghi P, Kawamura H, Anand S, Sato N, White LE, Hascall VC, Maytin EV. Hyaluronan suppresses epidermal differentiation in organotypic cultures of rat keratinocytes. Exp Cell Res. 2004;296: 123–134. 19. Weigel PH, Hascall VC, Tammi M. Hyaluronan synthases, J Biol Chem. 1997;272:13997–14000. 20. Stern R, Jedrzejas MJ. The hyaluronidases; their genomics,structures, and mechanisms of action. Chem Reviews. 2006;106:818–829. 21. Stern R. A new metabolic pathway: hyaluronan catabolism. Eur J Cell Biol. 2004;83(1–9):1–9.
Pigmentation
51 Hyperpigmentation in Aging Skin Tomohiro Hakozaki . Cheri L. Swanson . Donald L. Bissett
Introduction Human skin color varies greatly around the globe, from very pale Celtic skin to very darkly pigmented skin in subSaharan African populations. Yet, all of these skin types can develop hyperpigmentary problems with aging, for example, postinflammatory hyperpigmentation (PIH), solar lentigos, and melasma. These problems occur widely in the human population, and the methods used to control them are of great interest, with particular desire to achieve uniformity of skin color. Several proven targets for pigmentation control are known, but recent genomic and proteomic understanding of melanogenesis, the melanocyte, melanocyte–keratinocyte interaction, and melanocyte–fibroblast interaction has revealed potentially hundreds of proteins and other effectors involved in the pigmentation process. This body of knowledge, while complex, should provide the basis for understanding specific aberrations that lead to hyperpigmentary problems. Advanced laboratory screening models and tools for skin color quantification are also available. These are increasing the pace of screening of materials and clinical evaluation for their effectiveness. This brief review will focus on problems of hyperpigmentation (particularly as they apply to aging skin), investigative methods to measure and understand the problems, and topical cosmetic treatment approaches.
Pigmentation Process The pigmentation process has been extensively described in many other documents [1] and so will not be discussed in detail here. Briefly, melanocytes are specialized dendritic cells interspersed among basal keratinocytes and serve the primary function of producing melanin in intracellular organelles called melanosomes that are then distributed to surrounding keratinocytes. Each melanocyte is in contact with and distributes melanosomes to many keratinocytes via their dendritic processes. Melanins are complex polymers derived from tyrosine and other
intermediates, which are converted through a multistep process of oxidative and complexation reactions to brownblack eumelanins and yellow-red pheomelanins, which create the diversity of coloration observed across the human population. The regulation of melanin production is very complex and involves more than 80 genes [2, 3]. The synthesis is regulated by various extracellular signaling components that trigger a signal transduction cascade. There is also evidence that fibroblasts participate in this signaling [4]. While the baseline state of melanin in each individual’s skin is dictated by genetic composition, internal and external triggers such as aging and UV exposure can lead to significant alterations in net synthesis of the melanins [5].
Skin Changes with Aging Relevant to Pigmentation Over the course of an individual’s life, skin undergoes many changes [6], and there are many theories regarding the causes of the changes. While there is still much to be learned about these and probably other causes, it is clear that key influencers in hyperpigmentation are environmental effects and hormonal changes, which will be discussed in the following section in the context of specific hyperpigmentary disorders (see Section on Hyperpigmentary Disorders and Their Causes). In general, the number of active melanocytes per unit area of skin decreases with age (10–20% decline per decade), and there are more active melanocytes in chronically sun-exposed skin than in nonexposed skin [6]. This increased number of active melanocytes in sun-damaged skin indicates the influence of chronic UV exposure (e.g., on face, hands, and arms) in stimulating melanogenic potential. Since chronic UV exposure also alters dermal fibroblast function in aging skin and since fibroblasts appear to play a regulatory role in melanin production [4], dermal damage from sunlight may contribute to the production of hyperpigmentation in exposed aging skin.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_51, # Springer-Verlag Berlin Heidelberg 2010
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Hyperpigmentary Disorders and Their Causes Postinflammatory Hyperpigmentation (PIH) Skin insults that result in inflammation can induce postinflammatory hyperpigmentation [7], which is particularly evident in people with darker skin. Among such insults are acne lesions, ingrown hairs, scratches, insect bites, and surfactant damage. As an example of the latter, exposure of human forearm skin to the harsh surfactant sodium lauryl sulfate (SLS) under patch for a few hours will produce erythema within a day. Over the course of 1–2 weeks after this SLS exposure, hyperpigmentation will result, particularly in darker skin, but it will occur even in Caucasian skin. Topical treatment with antiinflammatory agents such as phytosterol will prevent this (> Table 51.1). The most common cause of hyperpigmentation (sunlight exposure of skin) is probably a postinflammatory response to UV damage to skin [8]. That response may be the result of an obvious acute inflammatory event such as sunburn or of repeated sub-erythemal exposures to UV. While in the latter, there may not be visible erythema, histologically, such exposed skin has elevated inflammatory cell content, yielding a ‘‘subclinical’’ inflammatory process. It is supported by the fact that topical treatment with anti-inflammatory agents immediately after UVB exposure prevents induction of delayed tanning [9]. Inflammation may result in hyperpigmentation through several mechanisms. Among them is direct stimulation of melanocytes by inflammatory mediators such as IL-1-alpha or ET-1 [10]. Reactive oxygen species such . Table 51.1 Postinflammatory hyperpigmentation (PIH) on the forearm Erythema grade (day 2)
PIH grade (day 11)
Vehicle
2.09
0.93
5% Phytosterola
1.71*
0.55*
Test agent
A 20% solution of sodium lauryl sulfate (SLS) was applied to the forearm skin of Caucasian subjects (n = 19) under occlusive patch (0.2 mL solution in a 19-mm diameter chamber patch). The patch was removed after 1–4 h, depending on the individual subject responsiveness. After washing the site to remove surface SLS, the skin was treated topically twice daily for 5 days with test agent. The skin was graded (0–4 grading scales) daily for erythema and pigmentation (postinflammatory hyperpigmentation; PIH) for 11 days (D. L. Bissett, unpublished work) a Phytosterol is a plant oil-derived mixture of stigmasterol, sitosterol, campesterol, and brassicasterol * Statistically significantly different (p < 0.05) versus vehicle
as superoxide and nitric oxide generated in damaged skin (e.g., from UV exposure) or released as by-products from inflammatory cells are also known stimulators of melanocytes. Additionally, damage induced to epidermal cells can lead to release of endocrine inducers of pigmentation such as alpha-MSH [11]. The resulting hyperpigmentation induced by all these effects provides some measures of protection against subsequent insult since melanin has both UV absorption and reactive oxygen species scavenging capacity. The melanin produced during an inflammatory event also can enter the dermis where it is engulfed by macrophages, producing ‘‘melanophages.’’ These cells are often retained in the upper dermis for prolonged periods since removal of dermal melanin apparently is a very slow process. Thus, postinflammatory hyperpigmentation can be a very long-lived problem for the skin [1].
Solar (Actinic) Lentigos These hyperpigmented spots are also known as lentigines, age spots, and liver spots. They occur on sun-exposed parts of the body (in particular, the hands, arms, face, upper chest, and shoulders) and thus occur due to chronic exposure of skin to UV and the resultant chronic inflammation, such as the epidermal endothelin cascade [10]. Their dark appearance certainly results from excessive melanin in the region, and may result from overproduction of melanin in the hyperactive melanocytes [12], longer retention of melanin in aging epidermis due to the slower turnover of this tissue layer [6], longer retention of melanin in keratinocytes within rete ridges [13], and dermal melanin-containing melanophages, which have been observed histologically to lie beneath the lentigines [1]. Since with aging, there is reduced wound healing [6, 14] and reduced clearance of materials from dermis apparently due to vascular and lymphatic changes [6], the residence time of melanophages in dermis may be very long. Within lesional lentigo skin, the rete ridges are greatly exaggerated, extending deeper into the dermis [12]. This deep penetration runs counter to the general observation of flattening of the convoluted dermal–epidermal junction with aging, evidenced by the diminution of the rete ridges [6]. In solar lentigenes, the basement membrane is also perturbed [12], which likely contributes to melanin entering the dermis to result in melanophage formation. These observations suggest that there has been a change in the genetic and phenotypic expressions of cells (perhaps both epidermal and dermal) within the spot area as compared to cells in the surrounding non-spot skin.
Hyperpigmentation in Aging Skin
The expression levels of several melanogenesis-associated genes are increased in actinic lentigos [15, 16]. There is also an accentuation of the epidermal endothelin inflammatory cascade [10], together with decreased proliferation and differentiation of lesional keratinocytes [17]. Many of these changes appear to be permanent since these spots persist even when further UV exposure is avoided. The details of these apparent genomic expression changes have not been defined. While lentigos appear to be permanent, their melanin content and thus their intensity will vary seasonally. For example, in evaluation of women with facial hyperpigmented spots in October versus December (in Kobe, Japan, or Cincinnati, Ohio, USA), there is a marked reduction in the size of spots over that time period, suggesting that the lack of continued exposure to sunlight in winter leads to gradual reduction in melanin production (seasonal fading) even in hyperpigmented spots [18, 19]. Additionally, in a separate examination of facial spots in March versus May (in Cincinnati, Ohio, USA), there was a marked increase in the size of spots [20], consistent with the expected increased pigmentation due to increased sun exposure in spring (seasonal darkening). From a consumer appearance standpoint, hyperpigmented spots and uneven pigmentation are important in the perception of age. In a series of studies [21], facial images were digitally modified to remove all age-defining textural features (e.g., facial furrows, folds, lines, wrinkles), leaving only pigmentation as the variable. Naı¨ve judge evaluation and computer image analysis of the images revealed that pigmentation features can contribute to up to 20 years in perceived age of individuals. So pigmentation is an important component of age perception.
Melasma The hyperpigmentary disorder melasma is not well understood [1]. It occurs typically as symmetrical lesions on the face, primarily in darker skin type females at puberty or later in life. Sunlight exposure is likely a factor in the development of melasma since it occurs on the face (a sun-exposed body site) and since the condition worsens in the summer. Most melasma sufferers have a hypersensitivity to ultraviolet radiation, i.e., they display a lower minimum erythemal dose, and even brief exposures to sunlight can stimulate hyperpigmentation. There is also a hormonal component, likely progesterone, since episodes of melasma are often associated with pregnancy and the use of hormonal birth control. There may also be an estrogen component since estrogen receptor expression is increased in melasma [22].
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In melasma lesions, there is excess melanin present in both the epidermis and upper dermis, associated with extravascular macrophages [1]. Since there is only a slight increase in the number of melanocytes, the abnormality appears to be in function of the skin cells, in particular, increased expression of factors in keratinocytes, fibroblasts, and melanocytes of the involved skin [23]. In contrast to PIH, there is no apparent inflammatory phase involved in its development. Additionally, there is likely a genetic component predisposing individuals to melasma, although the specific genetic basis for it is not defined.
Genomics and Proteomics of Pigmentation The pigmentation process is complex as evidenced particularly by recent genomic and proteomic analysis. There are approximately 1,500 gene products (proteins) expressed in melanosomes of all developmental stages, with 600 of them being expressed at any given time, and with 100 of them apparently unique to the melanosome [24]. Added to this are many other proteins (membrane-associated, cytoskeletal, transport, etc.) involved in pigmentation in both the melanocyte and the keratinocyte, indicating the complexity of the pigmentary process. While the basic process (e.g., stimulation of melanocytes and conversion of tyrosine to melanin) is well studied, there are many regulatory elements that have emerged from recent research involved in signaling, in the transport of melanosomes within the melanocyte, and the transfer of melanosomes to the keratinocyte [25]. This complexity merely offers a plethora of opportunities to understand the pigmentation process and to control it. Less well studied are the events that occur in the keratinocyte once melanosomes have been transferred there. In addition to the melanosome engulfment process itself, presumably there are intracellular signals, regulatory elements, and transport mechanisms to distribute the melanosomes within keratinocyte. There is a process of melanin degradation to produce ‘‘melanin dust,’’ an apparently enzymatic process that is more active in lighter skin versus darker skin individuals [26]. This is an area ripe for further study.
Pigmentation Control Agents As noted above, since there are many processes and proteins involved in the pigmentary process, there is a wide array of targets against which to screen for pigmentation control agents. Among the many targets [1] are inhibitors
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of melanocyte stimulation (e.g., antioxidants, anti-inflammatory agents), cell receptor antagonists (e.g., alpha-MSH antagonists), inhibitors of melanin synthesis enzymes (e.g., tyrosinase, TRP-1, TRP-2), inhibitors of melanosome transport within the melanocyte and transfer to the keratinocyte (e.g., PAR-2 antagonists), and activators of melanin degradation within the keratinocyte. While there are several potent drug, over the counter, and surgical approaches to control of pigmentation (e.g., hydroquinone, trans-retinoic acid, corticosteroid, chemical peel surgery, laser surgery, and combinations of these therapies), the discussion here will focus on agents used in cosmetic formulations [27]. A classic target is inhibition of tyrosinase, the first enzyme in the conversion of tyrosine to melanin. A wide array of compounds, such as kojic acid, arbutin, ascorbic acid, ellagic acid, sulfhydryl compounds, and resorcinols, are effective tyrosinase inhibitors, as is a more recently discussed deoxyarbutin [28]. However, since several of these materials also have other effects, it is difficult to directly connect a specific mechanism to the observed effect on pigmentation. For example, sulfhydryl compounds are also effective antioxidants. > Table 51.2 overviews a short list of the many possible targets and a few agents effective against them. In a recent work, niacinamide and glucosamine (in particular, its derivative N-acetyl glucosamine [NAG]) have been determined to be effective in reducing melanin production in culture. In vitro, glucosamine reduces production of melanin by inhibiting activation of tyrosinase [19], while niacinamide inhibits melanosome transfer from melanocytes to keratinocytes [18]. Cosmetic moisturizer formulations containing niacinamide alone are effective in reducing the appearance of hyperpigmented spots in vivo [18, 29] and the addition of NAG to the formula yields greater effectiveness [19] (> Fig. 51.1). Another new addition to the array of pigmentation control agents is N-undecylenoyl-L-phenylalanine, which has been reported to inhibit binding of alpha-MSH to the melanocyte in vitro and is effective as a component of cosmetic moisturizer formulations in clinical testing [30], as shown in > Fig. 51.2. Sunscreen is also effective in reducing the appearance of hyperpigmentation by preventing the entrance of UV into skin to stimulate melanocytes. Clinical testing among Japanese females in late summer-fall season (in Kobe, Japan) using SPF 15 sunscreen alone demonstrated acceleration of fading of facial tanning compared to the control [18]. However, even relatively high sunscreen dose (SPF 15) is not completely protective, such as against incidental sunlight exposure. In clinical testing involving daily use of
. Table 51.2 Pigmentation control targets and some reported effective agents Pigmentation control target examples
Effective agent examples
Tyrosinase inhibition
Hydroquinone, resorcinols, kojic acid, arbutin, deoxy-arbutin, ascorbic acid (vitamin C)
Tyrosinase copper chelation
Ellagic acid, kojic acid
Inhibition of tyrosinase glycosylation
Glucosamine, N-acetyl glucosamine, tunicamycin
Melanosome transfer
Niacinamide, protease inhibitors
Inhibit binding of alpha- N-undecylenoyl-phenylalanine MSH to melanocyte Down regulation of tyrosinase
Retinoid (trans-retinoic acid, retinol and its esters, retinaldehyde)
Antioxidant
Vitamin C compounds, vitamin E, sulfhydryl compounds
Anti-inflammatory agent
Hydrocortisone, phytosterol, glycyrrhetinic acid, tranexamic acid, chamomile extract
Increase epidermal turnover
Retinoids, salicylic acid, alpha-hydroxy acids, alphaketo acids, adenosine monophosphate
SPF 15 sunscreen [20], there was still a marked increase in the size of spots in March versus May (in Cincinnati, Ohio, USA), consistent with increased pigmentation due to increased sun exposure in spring (seasonal darkening). There was greater protection against this seasonal darkening when subjects used a three-way combination of SPF 15 sunscreen, niacinamide, and NAG, thus indicating the opportunity for greater effectiveness by combining sunscreen with non-sunscreen technologies.
Other Skin Chromophores While the focus of this review is on hyperpigmentation, it is informative to mention briefly that the appearance of pigmentation also involves other chromophores in the skin. For example, increased vascular content (hemoglobin) associated with hyperpigmentation has been described for melasma [31], and the same may be the case for hyperpigmented spots. Additionally, there is the spontaneous Maillard reaction (glycation; reaction between
Hyperpigmentation in Aging Skin
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. Figure 51.1 Hyperpigmented spot reduction by topical treatment with formulas containing niacinamide and N-acetyl glucosamine. Spot area fraction was determined by algorithm-based computer image analysis of Caucasian facial digital images (n = 35). More negative numbers indicate reduction in hyperpigmentation (improvement). N = niacinamide. NAG = N-acetyl glucosamine. The indicated p value for N + NAG is versus N
. Figure 51.2 Hyperpigmented spot reduction by topical treatment with formulas containing niacinamide and N-undecyloyl-Lphenylalanine. Spot area fraction was determined by algorithm-based computer image analysis of Japanese facial digital images (n = 40). More negative numbers indicate reduction in hyperpigmentation (improvement). N = niacinamide. NUP = N-undecylenoyl-L-phenylalanine. The indicated p value for N + NUP is versus N
protein and sugar) that produces yellow-brown chromophores [29, 32]. When this occurs in proteins with long biological half-lives (e.g., structural proteins), the glycation end products accumulate with aging, and the yellow-brown color increases and is persistent. Thus, hyperpigmented areas may appear to be darker in part due to other chromophores, opening the opportunity for further understanding of the problem and for additional approaches to treatment.
Investigative Tools Many of the targets noted above can be investigated in simple mechanism-specific solution assays, melanocyte cell culture, or melanocyte–keratinocyte coculture systems in the laboratory [18]. These methods permit screening of potentially large numbers of compounds for their inhibitory and stimulatory effects on the specific processes being evaluated. For example, one screening
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assay involves a simple mixture of tyrosinase and tyrosine in which a brown product (melanin) quickly appears and can be quantified colorimetrically. Tyrosinase inhibitors, of course, reduce the melanin produced. A simple assay of this type can be readily performed in a 96-well plate, allowing rapid robotic high-throughput screening of thousands of compounds. Assays involving cells are more complex, but even those can be constructed in a multi-well-plate format for moderate-throughput screening of potentially hundreds of compounds. Establishing an array of such simple assays permits the screening of a substantial library of compounds through all these assays to identify promising candidates quickly. Another useful laboratory model that has emerged over the past few years is the skin equivalent culture [4]. These three-dimensional cultures can contain both dermal and epidermal compartments, with fibroblasts, keratinocytes, melanocytes, and potentially other cell types. When they are raised to the air–liquid interface, the keratinocytes will be differentiated to form a stratum corneum structure. Also, live human skin organ culture has potential as a useful model [33]. Versus submerged culture of cells, an advantage of both skin equivalent and organ cultures is that they can be treated topically with simple solutions or even complex emulsion formulations or commercial skin care products. Another particular advantage is that they are not mechanism-specific – since most or all of the pigmentation machineries are present in the cultures, they potentially can be responsive to materials that affect any of the pigmentation targets. These cultures are available from commercial suppliers. While these are useful tools for material evaluation, they are relatively low throughput due particularly to the high cost of the cultures, and also to the time involved in manipulating the cultures over the course of the multiday experiments. There are some in vivo laboratory models that have been used to evaluate pigmentation inhibitory materials. As a recent example, zebra fish have been used in testing topical compounds [34]. Also, a mouse strain develops hyperpigmented spots in response to UV exposure [35], so has potential use as a lentigo model. Other models [1] include live human skin transplanted onto nude mice, Yucatan mini-pig, pigmented SKH-hr/2 mouse, and pigmented guinea pig. Like the skin-equivalent cultures, these models will not be mechanism-specific and thus are likely to be responsive to a wide range of material mechanisms. While all these models may not be broadly available, there is certainly an opportunity to pursue in vivo modeling as a tool in addressing pigmentation problems.
The final proof of value of a technology, of course, requires progressing materials from the laboratory into clinical testing to demonstrate on-skin activity. Clinical methods include live expert grading, chromameter [36], and color image capture and analysis [29]. A new useful clinical measurement tool in assessing effectiveness is based on the principles of noncontact SIAscopyTM, a recently described method to measure skin melanin content and distribution [37]. It rapidly captures facial maps of skin chromophores, permitting determination of the content and distribution of melanin in any spot or any area of the skin. It will also capture maps for any chromophore, and as long as the absorption spectra of the chromophores are known (e.g., hemoglobin, collagen), it can differentiate them to yield chromophore-specific distribution maps. Thus, there is also potential to determine spectra for other chromophores (glycated protein) and obtain distribution maps for them. Clinical testing on various body sites such as forearm, face, chest, and back [29, 38] have been reported, and all have utility in evaluating technology. Any thoroughly controlled clinical evaluation is expensive and therefore practicality limits testing to only the most promising candidates, several of which (as noted above) have proven to be effective in hyperpigmentation control.
Conclusion The continually increasing understanding of the pigmentation process and the underlying problems in hyperpigmentary conditions provide bases for establishing targets against which to screen new compounds to identify those that may be effective pigmentation control agents. With the advances in laboratory and clinical methodology, the screening process can occur much faster than in the past. In addition, the consumer desires effective pigmentation control technology, particularly in the cosmetic arena, since hyperpigmentation problems increase perceived aged. Advanced pigmentary system understanding and new research capabilities are setting the stage for future technological advancements.
Cross-references > Pigmentation > The
in Ethnic Groups New Face of Pigmentation and Aging
Hyperpigmentation in Aging Skin
References 1. Nordlund JJ, Boissy RE, Hearing VJ, King RA, Ortonne JP. The Pigmentary System. New York: Oxford University Press, 1998. 2. Hearing VJ. Biochemical control of melanogenesis and melanosomal organization. J Invest Dermatol Symp Proc. 1999;4:24–28. 3. Schallreuter KU. Advances in melanocyte basic science research. Dermatol Clin. 2007;25:283–291. 4. Cario-Andre M, Pain C, Gauthier Y, et al. In vivo and in vitro evidence of dermal fibroblasts influence on human epidermal pigmentation. Pig Cell Res. 2006;19:434–442. 5. Costin GE, Hearing VJ. Human skin pigmentation: melanocytes modulate skin color in response to stress. FASEB J. 2007;21:976–994. 6. Gilchrest BA. Skin and Aging Processes. Boca Raton: CRC Press, 1984. 7. Taylor SC. Cosmetic problems in skin of color. Skin Pharmacol Appl Skin Physiol. 1999;12:139–143. 8. Hachiya A, Kobayashi T, Takema Y, et al. Biochemical characterization of endothelin-converting enzyme-1 alpha in cultured skinderived cells and its postulated role in the stimulation of melanogenesis in human epidermis. J Biol Chem. 2002;277:5395–5403. 9. Takiwaki H, Shirai S, Kohno H, et al. The degrees of UVB-induced erythema and pigmentation correlate linearly and are reduced in a parallel manner by topical anti-inflammatory agents. J Invest Dermatol. 1994;103:642–646. 10. Kadono S, Manaka I, Kawashima M, et al. The role of the epidermal endothelin cascade in the hyperpigmentation mechanism of lentigo senilis. J Invest Dermatol. 2001;116:571–577. 11. Imokawa G. Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders. Pig Cell Res. 2004;17: 96–110. 12. Noblesse E, Nizard C, Cario-Andre M, et al. Skin ultrastructure in senile lentigo. Skin Pharmacol Physiol. 2006;19:95–100. 13. Cario-Andre M, Lepreux S, Pain C, et al. Perilesional vs. lesional skin changes in senile lentigo. J Cutan Pathol. 2004;31:441–447. 14. Makrantonaki E, Zouboulis CC. Molecular mechanisms of skin aging: state of the art. Ann N Y Acad Sci. 2007;1119:40–50. 15. Motokawa T, Matsunaga J, Tomita Y. Messenger RNA levels of melanogenesis-associated genes in lentigo senilis lesions. J Dermatol Sci. 2005;37:120–123. 16. Unver N, Freyschmidt-Paul P, Horster S, et al. Alterations in the epidermal-dermal melanin axis and factor XIIIa melanophages in senile lentigo and ageing skin. Br J Dermatol. 2006;155:119–128. 17. Aoki H, Moro O, Tagami H, et al. Gene expression profiling analysis of solar lentigo in relation to immunohistochemical characteristics. Br J Dermatol. 2007;156:1214–1223. 18. Hakozaki T, Minwalla L, Zhuang J, et al. The effect of niacinamide on reducing cutaneous pigmentation and suppression of melanosome transfer. Br J Dermatol. 2002;147:20–31. 19. Bissett DL, Robinson LR, Raleigh PS, et al. Reduction in the appearance of facial hyperpigmentation by topical N-acetyl glucosamine. J Cosmet Dermatol. 2007;6:20–26. 20. Kimball AB, Kaczvinsky JR, Bissett DL, et al. Reduction in the appearance of facial hyperpigmentation by a combination of
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topical niacinamide plus N-acetyl glucosamine: results of a randomized, double-blind, placebo-controlled trial. Br J Dermatol, submitted. Fink B, Matts PJ. The effects of skin colour distribution and topography cues on the perception of female facial age and health. J Eur Acad Dermatol Venereol. 2008;22:493–498. Lieberman R, Moy L. Estrogen receptor expression in melasma: results from facial skin of affected patients. J Drugs Dermatol. 2008;7:463–465. Kang HY, Hwang JS, Lee JY, et al. The dermal stem cell factor and c-kit are overexpressed in melasma. Br J Dermatol. 2006;154:1094–1099. Chi A, Valencia JC, Hu Z-Z, et al. Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes. J Proteome Res. 2006;5:3135–3144. Boissy RE. Melanosome transfer to and translocation in the keratinocyte. Exp Dermatol. 2003;12(S2):5–12. Chen NN, Seiberg M, Lin CB. Cathepsin L2 levels inversely correlate with skin color. J Invest Dermatol. 2006;126:2345–2347. Nakayama H, Ebihara T, Satoh N, et al. Depigmentation agents. In: Elsner P, Maibach HI (eds) Cosmeceuticals and Active Cosmetics. Boca Raton: Taylor & Francis, 2005. Boissy RE, Visscher M, DeLong MA. Deoxyarbutin: a nevel reversible tyrosinase inhibitor with effective in vivo skin lightening potency. Exp Derm. 2005;14:601–608. Bissett DL, Miyamoto K, Sun P, et al. Topical niacinamide reduces yellowing, wrinkling, red blotchiness, and hyperpigmented spots in aging facial skin. Int J Cosmet Sci. 2004;26:231–238. Bissett DL, Robinson LR, Raleigh PS, et al. Reduction in the appearance of facial hyperpigmentation by topical N-undecyl-10-enoyl-Lphenylalanine and its combination with niacinamide. J Cosmet Dermatol, submitted. Kim EH, Kim YC, Lee ES, et al. The vascular characteristics of melasma. J Dermatol Sci. 2007;46:111–116. Dyer DG, Dunn JA, Thorpe SR, et al. Accumulation of maillard reaction products in skin collagen in diabetes and aging. J Clin Invest. 1993;91:2463–2469. Backvall H, Wassberg C, Berne B, et al. Similar UV responses are seen in a skin organ culture as in human skin in vivo. Exp Dermatol. 2002;11:349–356. Choi T-Y, Kim J-H, Ko DH, et al. Zebrafish as a new model for phenotype-based screening of melanogenic regulatory compounds. Pig Cell Res. 2007;20:120–127. Furuya R, Akiu S, Ideta R, et al. Changes in the proliferative activity of epidermal melanocytes in serum-free primary culture during the development of ultraviolet radiation B-induced pigmented spots in hairless mice. Pig Cell Res. 2002;15:348–356. Alaluf S, Atkins D, Barrett K, et al. The impact of epidermal melanin on objective measurements of human skin colour. Pig Cell Res. 2002;15:119–126. Matts PJ, Dykes PJ, Marks R. The distribution of melanin in skin determined in vivo. Br J Dermatol. 2007;156:620–628. Ravnbak MH, Philipsen PA, Wiegell AR, et al. Skin pigmentation kinetics after UVB exposure. Acta Dermatol-Venereol. 2008;88: 223–228.
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49 In vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model Akira Date . Tomohiro Hakozaki
Introduction When the skin is exposed to ultraviolet (UV) light consisting of UVA (320–400 nm) and UVB (290–320 nm), reactive oxygen species (ROS) such as superoxide anion radical (O2–), hydrogen peroxide (H2O2), hydroxyl radical (OH), singlet oxygen (1O2), as well as lipid peroxides, and their radicals (LOOH and LOO) are formed [1, 2]. It is well documented that these free radicals and ROS cause oxidative cellular stress, cell injury, and DNA damage in the epidermis [3, 4], and eventually induce inflammation, skin photoaging, phototoxicity, or malignant tumors [5–8]. To protect skin from these radical species, there are multiple natural defense mechanisms in the skin. For instance, antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) play important roles in protecting the skin against degenerative changes by free radicals or ROS [9, 10]. In order to examine the impact and role of UVinduced free radicals or ROS in the skin, it is essential to detect and visualize them in a real time. However, due to extremely short-lived and essentially nonemissive nature, free radials and ROS are difficult to detect directly. Several evaluation methods such as chemiluminescence (CL) probe [1, 11], photon emission detection [12–15], fluorescence detection [16, 17], and electron spin resonance (ESR) spectroscopy using spin probes [18–21] have been developed to detect ROS and investigate their species and behaviors. Meanwhile, in the past 2 decades, human skin equivalent models have been developed for multiple purposes, such as a replacement of animal models for compound safety evaluation. Now several models have become commercially available, and are used not only for safety assessment, but also to investigate biological functions of skin or responses against various stimulations such as compound treatment or UV exposure.
This chapter introduces recent research and methods to detect and visualize oxidative stress or ROS utilizing human skin equivalent models.
Oxidative Stress Measurement for the Evaluation of UV-Induced DNA Damage in a Human Skin Equivalent Model Several techniques were developed for assessing skin damages in human skin equivalent models. Among them, an oxidatively modified DNA base, 8-hydroxy-2’-deoxyguanosine (8-OHdG), is induced by hydroxyl radical (OH), singlet oxygen (1O2), photodynamic reaction, or peroxynitrite (ONOO ). It is also mutagenic when present during DNA replication [22]. In 2006, a combination of a human skin equivalent model and 8-OHdG immunohistochemistry was proposed by Toyokuni et al. [23]. UVBinduced DNA modification was evaluated by utilizing produced 8-OHdG level as a biomarker. Specifically, the human skin equivalent model was exposed to UVB radiation, and the induction of 8-OHdG was examined by immunohistochemical analysis with catalyzed signal amplification on formalin-fixed paraffin sections. The immunohistochemical images were processed with Image J (National Institutes of Health [NIH] image software) to qualify 8-OHdG by multiplying positively stained area and density (8-OHdG index) [4, 24]. Formation of 8-OHdG in the skin equivalent model by UVB exposure was demonstrated, and it is produced in a UV-dose-dependent manner (> Fig. 49.1). Interestingly, little nuclear staining of 8-OHdG was observed in the negative control samples without UVB exposure, but the increase of 8-OHdG staining was clearly observed in the UVB-exposed samples. The effect of pretreatment of several antioxidants was also examined. The formation of 8OHdG was effectively suppressed by the treatment of antioxidative compounds (> Fig. 49.2). It provides evidence to
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_49, # Springer-Verlag Berlin Heidelberg 2010
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In vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model
. Figure 49.1 Localization of 8-OHdG in a human skin equivalent model. (a) Hematoxylin and eosin staining of an untreated specimen. Immunostaining of 8-OHdG after UVB exposure (2.25 mJ/cm2/min) at various time periods. (b) 0 min, (c) 12 min, (d) 18 min, (e) 24 min, (f) 36 min, (g) 48 min, and (h) 60 min. Bar = 100 mm
the fact that the combination of human skin equivalent model and 8-OHdG immunohistochemistry is considered as a distinct screening strategy for identifying active compounds, which prevent UV-induced skin damage with the following three characteristics: (1) alternative to animal experiments; (2) expensive instruments are not required; and (3) higher sensitivity compared with the animal model. More recently, new research was reported by Bernerd and Asselineau utilizing a skin reconstructed model containing a dermal equivalent and a fully differentiated epidermis [25]. The authors investigated the effects of UV light (UVB and UVA) on photo-damage by employing the sunburn cell formation, which is linked to the presence of DNA lesions, such as pyrimidine dimers and (6,4) photoproducts being a direct chromophore for UVB radiation. They found that UVB-induced damage was
essentially epidermal, with the typical sunburn cells and DNA lesions, whereas UVA-induced damage was mostly located within the dermal compartment. The model and end points used for UVB- and UVA-induced damages appeared to be very useful for the in vitro evaluation of sunscreens or compounds, in particular to investigate their protective effects against the effects of UV radiation. It will also allow to distinguish the efficiency of UV absorbers depending on their absorption spectrum [25, 26]. Currently, there is an emerging concept that there are fragile genomic sites to oxidative stress as well as UVspecific DNA base modifications. In the future, human skin equivalent models will be further applied to the investigation of skin damages at molecular or cellular level by combining with the emerging new technologies such as laser capture micro-dissection (LCM), which is a
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. Figure 49.2 Amelioration of UVB-induced 8-OHdG in a human skin equivalent model by various agents. Immunohistochemistry of 8-OHdG after UVB exposure of 40.5 mJ/cm2 (18 min) (a) with or (b) without prior treatment of 10 mM ascorbate. (c) Quantitation of 8-OHdG immunostaining by NIH image freeware. Asc, ascorbate; bC, b-carotene; SOD, Cu, Zn-superoxide dismutase. Means SEM, N = 5; **p < 0.01 vs untreated control (No UV); #p < 0.05, ##p < 0.01 versus UVB exposure alone. (d) Cell viability determined by MTT assay after UVB exposure of 135 mJ/cm2 (60 min). Means SEM, N = 4 6; **p < 0.01 vs UVB exposure alone. Bar = 100 mm
new method for isolating specific cells of interest from microscopic regions of tissue that has been sectioned [27].
Photon Emission and Fluorescence Technique to Detect ROS in Human Skin Equivalent Models Photons participate in many atomic and molecular interactions and processes. Recent biophysical research has discovered ultraweak radiation emitted from the biological tissues. Several physical or chemical environmental stressors generate ROS, which trigger oxidative reactions in/around the cells or tissues and thereby induce a correlated ultraweak photon emission (UPE) signal. Several works on photon emission detection and imaging in the skin have been
documented just recently. In 2008, Khabiri and Hangen reported a highly sensitive method to assess oxidative processes in biological molecules using weak photon emission generated due to oxidation of proteins or amino acids. For instance, strong UPE signals are detected by the oxidation of Phe, Trp, His, and Cys, and weak UPE signals from Lys and Thr. They proposed the noninvasive method for monitoring of UVA-induced oxidative skin stress by UPE measurement to assess the potency of topical antioxidants in ex vivo porcine skin and in vivo human skin [12, 13]. Niggli et al. reported an improved UPE measurement from UVA laser-induced biophotonic emission of different cultured cells to detect biophysical changes between young and adult fibroblasts, as well as the changes between fibroblasts and keratinocytes [14]. Van Wijk et al. recently reviewed the current status of human photon emission
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techniques and the protocols for recording the human oxidative status. Systematic studies on human emission have presented information on: (a) procedures for reliable measurements and spectral analysis; (b) anatomic intensity of emission and left–right symmetries; (c) biological rhythms in emission; (d) physical and psychological influences on emission; (e) novel physical characteristics of emission; and (f) the identification of UPE with the staging of ROS-related damage and disease. It is concluded that both patterns and physical properties of UPE hold considerable promise as measure for the oxidative status [15]. While UPE detection is one emerging technique, fluorescence techniques with chemical probes for detection and visualization of ROS are also useful tools. Fluorescence as well as chemiluminescence can offer high detection sensitivity. There are several research studies on the detection and imaging of ROS in the UV-exposed human skin equivalent models by using fluorescent techniques. Hanson and Clegg reported the method to observe and quantify UV-induced ROS in ex vivo human skin [16]. Their proposed method consists of two-photon fluorescence microscopy to detect UV-induced ROS. They observed ROS by using a human skin equivalent model, and the epidermis and the dermis of ex vivo human skin. In their study, the human epidermal skin model was incubated with the nonfluorescent ROS probe dihydrorhodamine (DHR), which reacted with ROS such as O2 and H2O2 to form fluorescent rhodamine-123. They reported that the two-photon excitation provides a depth penetration through the skin unlike confocal microscopic techniques. Thus, this method can provide submicron spatial resolution such that subcellular areas where ROS are generated could be detected. This would enable the monitoring of UV-induced ROS at different depths within the skin. In the future, the method might be applicable to evaluate the ability of sunscreens or antioxidants to prevent ROS generation and photo-damage at targeted depth or the region in the skin [17].
Electron Spin Resonance Technique to Detect ROS in a Human Skin Equivalent Model Electron spin resonance (ESR) techniques have been widely used to study ROS and oxidative stress in biological systems in vitro. However, limited number of research studies has been documented by using human skin equivalent models, ex vivo human skin, and even less for in vivo human skin.
In 2000, Togachi et al. reported a spin-trapping detection of ROS using X-band ESR spectroscopy and described the detection of ROS such as singlet oxygen (1O2) and hydroxyl radical (OH) by the spin trap 5,5-dimethyl-1pyrroline-N-oxide (DMPO) and ESR spectroscopy in vitro. They also did in vivo ESR detection of ROS using a nitroxide spin probe, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCM), for noninvasive imaging of oxidative stress in living animals [18]. In 2003, Herrling et al. reported the detection of UVinduced free radicals and ROS generated in the ex vivo human skin (skin biopsies) by ESR spectroscopy using several nitroxides such as 2,2,6,6-tetramethylpiperidine1-oxyl (TEMPO), 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCM), and 3-carboxy-2,2,5,5-tetramethyl pyrrolidine-1-oxyl (PCA) [19]. The authors found that the reduction rates were different among the nitroxides. TEMPO was decreased due to both UV radiation and enzymatic activity in the skin, while PCM and PCA were sufficiently stable in the skin, and solely reduced by UVgenerated free radicals/ROS. They also imaged the spatial distribution of UV-induced free radicals and ROS by using the PCA probe. By assuming the homogeneous distribution of PCA in the skin, they estimated the penetration profile of UVA and UVB irradiation, as the UV irradiation decreases the PCA intensity corresponding to its irradiance and penetration into the skin. Interestingly, this reduction was caused mainly by UVA radiation (320–400 nm), suggesting the importance of UVA protection. In 2006, they reported their further work on detection and imaging of UVA-induced ROS in the human skin biopsies by the combination of the nitroxide spin probe PCA and an L-band ESR spectrometer. The main parts of ROS were generated by UVA (320–400 nm) so that the spatial distribution of free radicals reaches up to the lower side of the dermis. In addition, they proposed a new radical sun protection factor (RSF) to assess antioxidant compounds and UV filters on how much they can protect against UV-induced ROS [20]. As another application of ESR techniques, Date and Hakozaki’s group recently reported ESR spin-trapping method with new probes to detect ROS in a human epidermal skin model [21]. 5,5-Dimethyl-1-pyrolline-1-oxide (DMPO) probe, which is known to detect superoxide anion radical (O2–) and hydroxyl radical (OH) selectively was used. The combination of 2,2,6,6-tetramethyl4-pyperidone (TMPD) and K3Fe(CN)6 for the detection of singlet oxygen (1O2) was also examined. The model was verified by testing the application of the antioxidants known to scavenge specific ROS such as mannitol (OH scavenger), SOD (O2– scavenger), ascorbate (O2–, OH,
In vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model
and 1O2 scavenger), and b-carotene (1O2 quencher). It demonstrated diminishing of the ESR signal of DMPOOH (to detect O2– and OH) by mannitol (OH scavenger) or SOD (O2– scavenger), as well as suppression of the ESR signal of 4-oxo-TEMPO (TMPD-1O2) to detect 1 O2 by b-carotene (1O2 quencher) compared with UVBexposed control samples, as expected (> Figs. 49.3 and > 49.4). This technique will be a useful tool not only to predict UV-induced ROS-related responses in human skin, but also to find the protective compounds against UV-induced oxidative stress in the human skin.
Chemiluminescent Technique to Detect or Visualize ROS in a Human Skin Equivalent Model Chemiluminescent (CL) techniques have been widely used to study oxidative stress in biological systems in vitro; however, several methods are reported using human skin equivalent models, explant organ skin, or in vivo skin. In 2000, a unique technique using an in vivo real-time chemiluminescent (RT-CL) detection and two-dimensional ultra low-light imaging of endogenously generated ROS in the skin of living animals after UVA light exposure was proposed by Yasui and Sakurai [1]. They used a CL probe, cypridina luciferin analog (CLA) and an ultra-low-light
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imaging apparatus equipped with a charge-couple device (CCD) camera (NightOWL™). CLA reacts specifically with superoxide anion radical (O2–) or singlet oxygen (1O2), and emits chemiluminescence at 380 nm according to the consecutive reaction through the intermediate, dioxetane (> Fig. 49.5). The CL emissions are measured using the high-performance ultra-low-light imaging luminograph system. The authors applied this CL method to measure the rate constants of O2– or 1O2 and also demonstrated that it is useful not only in characterizing the ROS, but also in finding protective compounds against UV-induced skin damage and in characterizing ROS generated in the UV (UVA and UVB) light-exposed skin [2, 28]. With this method, they identified that superoxide anion radical was formed intrinsically, and superoxide anion radical and singlet oxygen were generated by UVA exposure to living animal skin. In addition, they indicated that antioxidative ability against ROS in the skin decreases by aging [2]. The former method is informative and useful; however, it uses live animals. In 2006, the combination of a human skin equivalent model (EpiDerm™ skin model EPI-200, MatTek) and the RT-CL method consisting of a sensitive CL probe CLA and an ultra-low-light imaging apparatus was proposed by Yasui et al. as a new novel tool to detect and visualize UVB-induced ROS generation [29]. With this system, CL emission due to the reaction
. Figure 49.3 Effect of antioxidants to suppress O2 and OH by ESR with DMPO. The human skin equivalent model was treated with 1 M DMPO, and exposed to UVB at a dose of 27 mJ/cm2. 10 mM Mannitol, 10 mM ascorbate, and 100 mM SOD pretreatments significantly scavenged the ESR signal of DMPO-OH, and 100 mM catalase (CAT), 10 mM glutathione (GSH), and 10 mM diethylenetriamine-N,N,N’,N’’,N’’-pentaacetic acid (DTPA) pretreatments slightly scavenged its ESR signal. While, 10 mMb carotene (BCA) and 10 bmg/mL chondroitin sulfate B (CSB) pretreatments did not scavenge its ESR signal **p < 0.01 vs UVB exposure (n = 3/each group)
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. Figure 49.4 Effect of antioxidants to suppress 1O2 measured by ESR with TMPD/ K3Fe(CN)6. The human skin equivalent model was treated with 200 mM TMPD and exposed to UVB at a dose of 27 mJ/cm2, and then 100 mM K3Fe(CN)6 was added to the system. The ESR signal of 4-oxo-TEMPO due to the reaction between 1O2 and TMPD was considerably observed. 10 mM ascorbate (ASC), 10 mM b-carotene (BCR), and 10 mM glutathione pretreatments significantly scavenged the ESR signal of 4-oxo-TEMPO (TMPD-1O2), and 100 mM catalase (CAT), 10 mg/mL chondroitin sulfate B (CSB),10 mM diethylenetriamineN,N,N’,N’’,N’’-pentaacetic acid (DTPA), 10 mM mannitol, and 100 mM SOD pretreatments slightly scavenged its ESR signal **p < 0.01 vs UVB exposure (n = 3/each group)
. Figure 49.5 Principle of CLA probe. Cypridina luciferin analog (CLA) reacts specifically with superoxide anion radical (O2–) or singlet oxygen (1O2) and emits chemiluminescence at 380 nm according to the consecutive reaction through the intermediate, dioxetane
of CLA with endogenously generated ROS increased significantly in the UVB-exposed skin compared with that in the intact skin, maximum level being observed at a dose of 27 mJ/cm2. The treatment of SOD (O2– scavenger) and b-carotene (1O2 quencher) effectively suppressed UVBinduced CL intensities, indicating the generation of O2– and 1O2 in the skin equivalent model under UVB exposure. These results were consistent with those observed in the skin of living animals, thus supporting the relevancy of usage of skin equivalent model for the purpose.
Date et al. have further evaluated various antioxidative compounds such as ascorbate, b-carotene, SOD, and yeast ferment filtrate (YFF), which was reported to suppress superoxide anion and hydroxyl radical [21]. A typical visualization of CL from the human skin equivalent model with and without UVB exposure is shown in > Fig. 49.6 [30]. The UVB-exposed skin samples exhibited significantly higher CL levels (red color) than did the intact skin samples (green-yellow color), indicating the increased generation of ROS in the epidermal skin model. The tested
In vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model
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. Figure 49.6 Typical visualization of the chemiluminescent signals in the human skin equivalent model due to ROS generation. The highest level (red color) of ROS generation in the skin was found 44.5 min after UVB exposure at the dose of 27 mJ/cm2. The low chemiluminescence (green-yellow color) was also observed in the human skin equivalent model without UVB exposure, suggesting that live epidermal cells produce ROS intrinsically. ROS reducing effects of antioxidative compounds such SOD, b-carotene, ascorbate, and YFF were clearly demonstrated
. Figure 49.7 Semiquantification of UVB-induced ROS and effect of antioxidants. Suppressive effects of SOD, b-carotene (BCR), ascorbate (ASC), and YFF on the UVB-induced ROS in the human skin equivalent model. Data are expressed as the means SD for 8–10 samples in each experiment. Significant suppressions versus control [UVB(+)] were observed in all treatments in UVB-exposed group (*p < 0.05), while no significant differences versus control [UVB( )] were found in all treatments in non-UVB-exposed group (p > 0.05)
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compounds greatly reduced UVB-induced CL intensities immediately after the measurement. For semiquantitative comparison, the areas under the curve of CL intensities (AUC) were calculated for all tested compounds as shown in > Fig. 49.7. Compared with the UVB-induced control group, the treatment with antioxidative compounds exhibited statistically significantly lower CL intensities. This unique method will be an effective tool for (1) investigating the impact of ROS in human skin damage and photoaging, and (2) screening protective compounds to suppress ROS generation against UVB-induced skin damage with a powerful visualization ability.
Conclusion With the advance of technology, more and more methods are available to detect and visualize free radicals and ROS in the skin and its equivalent models. In the future, these in vitro methods using human skin equivalent models will be considered to be a relevant and handy tool to identify protective compounds against oxidative stress and its aging effect, and predict ROS-related responses in human skin as a substitute for animal model.
Cross-references > Effect
of Ozone on Cutaneous Tissues and Genetic Factors in Facial Aging in
> Environmental
Twins > Global
Warming and its Dermatological Impact on Aging Skin > Skin Photodamage Prevention: State of the Art and New Prospects
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5. Kligman LH. The ultraviolet-irradiated hairless mouse: a model for photoaging. J Am Acad Dermatol. 1989;21:623–631. doi:10.1016/ 0738-081X(95)00154-8 6. Ichihashi M, Ueda M, Budiyanto A, Bito T, Oka M, Fukunaga M, Tsuru K, Horikawa T. UV-induced skin damage. Toxicology. 2003;189:21–39. doi:10.1016/S0300–483X(03)00150-1 7. Bech-Thomsen N, Wulf HC. Carcinogenic and melanogenic effects of a filtered metal halide UVA source and a tubular fluorescent UVA tanning source with or without additional solar-simulated UV radiation in hairless mice. Photochem Photobiol. 1995;62:773–779. doi:10.1111/j.1751-1097.1995.tb08729.x 8. Kripke ML, Cox PA, Alas LG, Yarosh DB. Pyrimidine dimmers in DNA initiate systemic immunosuppression in UV-irradiated mice. Proc Natl Acad Sci USA. 1992;89(16):7516–7520. PMID: 1502162 PMCID: PMC49741 9. Sasaki H, Akamatsu H, Horio T. Protective role of copper, zinc superoxide dismutase against UVB-induced injury of the human keratinocyte cell line HaCaT. J Invest Dermatol. 2000;114:502–507. doi:10.1046/j.1523-1747.2000.00914.x 10. Hellemans L, Corstjens H, Neven A, Declercq L, Maes D. Antioxidant enzyme activity in human stratum corneum shows seasonal variation with an age-dependent recovery. J Invest Dermatol. 2003;120:434–439. doi:10.1046/j.1523–1747.2003.12056.x 11. Ou-Yang H, Stamatas G, Saliou C, Kollias N. A chemiluminescence study of UVA-induced oxidative stress in human skin in-vivo. J Invest Dermatol. 2004;122:1020–1029. doi:10.1111/j.0022-202X. 2004.22405.x 12. Khabiri F, Hagens R, Smuda C, Soltau A, Schreiner V, Wenck H, Wittern KP, Duchstein HJ, Mei W. Non-invasive monitoring of oxidative skin stress by ultraweak photon emission (UPE)-measurement. I: mechanisms of UPE of biological materials. Skin Res Technol. 2008;14(1):103–111. doi:10.1111/j.1600-0846.2007.00205.x 13. Hagens R, Khabiri F, Schreiner V, Wenck H, Wittern KP, Duchstein HJ, MeiW. Non-invasive monitoring of oxidative skin stress by ultraweak photon emission measurement. II: biological validation on ultraviolet A-stressed skin. Skin Res Technol. 2008;14 (1):112–120. doi:10.1111/j.1600-0846.2007.00207.x 14. Niggli HJ, Tudisco S, Lanzano` L, Applegate LA, Scordino A, Musumeci F. Laser-ultraviolet-A induced ultra weak photon emission in human skin cells: A biophotonic comparison between keratinocytes and fibroblasts. Indian J Exp Biol. 2008;46(5):358–363. PMID: 18697620 15. Van Wijk R, Van Wijk EP, Wiegant FA, Ives J. Free radicals and lowlevel photon emission in human pathogenesis: state of the art. Indian J Exp Biol. 2008;46(5):273–309. PMID: 18697612 16. Hanson KM, Clegg RM. Observation and quantification of ultraviolet-induced reactive oxygen species in ex vivo human skin. Photochem Photobiol. 2002;76:7–63. doi:10.1562/0031-8655(2002) 0760057OAQOUI2.0.CO2 17. Hanson KM, Clegg RM. Two-photon fluorescence imaging and reactive oxygen species detection within the epidermis. Methods Mol Biol. 2005;289:413–422. ISBN: 9781592598304 PMID: 15502202 18. Togashi H, Shinzawa H, Matsuo T, Takeda Y, Takahashi T, Aoyama M, Oikawa K, Kamada H. Analysis of hepatic oxidative stress status by electron spin resonance spectroscopy and imaging. Free Radic Biol Med. 2000;28(6):846–853. doi:10.1016/S0891-5849(99)00280-4 19. Herrling T, Fuchs J, Rehberg J, Groth N. UV-induced free radicals in the skin detected by ESR spectroscopy and imaging using nitroxides. Free Radic Biol Med. 2003;35(1):59–67. doi:10.1016/S0891-5849(03) 00241-7
In vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model 20. Herrling T, Jung K, Fuchs J. Measurements of UV-generated free radicals/reactive oxygen species (ROS) in skin. Spectrochim Acta A: Mol Biomol Spectrosc. 2006;63(4):840–845. doi:10.1016/j. saa.2005.10.013 21. Date A, Hakozaki T, Yoshii T, Yasui H, Sakurai H. Detection and identification of reactive oxygen species and followed free radicals generated in the UVB-exposed three dimensional human epidermal cells- EpidermTM as measured by ESR spin-trapping method. In: The 126th annual meeting of pharmaceutical society of Japan, Sendai, Japan (2006), pp 28, [R] am-174. 22. Kasai H. Analysis of a form of oxidative DNA damage, 8-hydroxy-2’deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutat Res. 1997;387:147–163. PMID: 9439711 23. Toyokuni S, Yasui H, Date A, Hakozaki T, Akatsuka S, Kohda H, Yoshii T, Sakurai H. Novel screening method for ultraviolet protection: Combination of a human skin-equivalent model and 8-hydroxy-2’-deoxyguanosine. Pathol Int. 2006;56(12):760–762. doi: 10.1111/j.1440-1827.2006.02043.x 24. Toyokuni S, Tanaka T, Hattori T, Nishiyama Y, Yoshida A, Uchida K, Hiai H, Ochi H, Osawa T. Quantitative immunohistochemical determination of 8-hydroxy-2’-deoxyguanosine by a monochlonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model. Lab Invest. 1997;76:365–374. PMID: 9121119
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25. Bernerd F, Asselineau D. An organotypic model of skin to study photodamage and photoprotection in-vitro. J Am Acad Dermatol. 2008;58(5 Suppl 2):155–159. doi:10.1016/j.jaad.2007.08.050 26. Fourtanier A, Moyal D, Seite´ S. Sunscreens containing the broadspectrum UVA absorber, Mexoryl1 SX, prevent the cutaneous detrimental effects of UV exposure: a review of clinical study results. Photodermatol Photoimmunol Photomed. 2008;24(4):164–174. PMID 18717957 doi:10.1111/j.1600-0781.2008.00365.x 27. Espina V, Heiby M, Pierobon M, Liotta LA. Laser capture microdissection technology. Expert Rev Mol Diagn. 2007;7(5):647–657. doi:10.1586/14737159.7.5.647. PMID 17892370 28. Nishimura H, Yasui H, Sakurai H. Generation and distribution of reactive oxygen species in the skin of hairless mice under UVA: studies on in-vivo chemilumiminescent detection and tape stripping methods. Exp Dermatol. 2006;15(11):891–899. doi: 10.1111/j.16000625.2006.00484.x 29. Yasui H, Hakozaki T, Date A, Yoshii T, Sakurai H. Real-time chemiluminescent imaging and detection of reactive oxygen species in the UVB-exposed human skin equivalent model. Biochem Biophys Res Commun. 2006;347:83–88. doi:10.1016/j.bbrc.2006.06.046 30. Hakozaki T, Date A, Yoshii T, Toyokuni S, Yasui H, Sakurai H. Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model. Arch Dermatol Res. 2008;300(Suppl 1):S51–56. doi: 10.1007/s00403-007-0804-3
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42 Infrared A-induced Skin Aging Peter Schroeder . Jean Krutmann
Introduction Extrinsic skin aging has, for many years, been mainly attributed to ultraviolet (UV) radiation. Recently, it has become evident that other parts of the solar electromagnetic spectrum contribute as well. Among these, infrared radiation, especially Infrared A, has received increasing attention. This chapter will summarize the current knowledge about the epidemiological evidence, molecular principles, and prevention/protection, as it concerns skin aging induced by Infrared A.
Infrared Radiation Physical Basics and Natural and Artificial Sources Solar radiation is filtered by the earth’s atmosphere; the part reaching the earth surface includes the wavelengths from 290 to 4,000 nm and is divided into three bands: ultraviolet radiation (UV, 290–400 nm), visible light (400–760 nm), and infrared radiation (IR, 760–4,000 nm). Infrared radiation is further subdivided into IRA (l = 760–1,440 nm), IRB (l = 1,440–3,000 nm), and IRC (l = 3,000 nm–1 mm). While the photon energy of IR is lower than that of UV, the total amount of solar energy reaching human skin contains approximately 54% IR, while UV only accounts for 7% [1]. Most of the IR radiation lies within the IRA band (30% of total solar energy), which deeply penetrates the human skin, while IRB and IRC only affect the upper skin layers (> Fig. 42.1). In comparison, IRA penetrates better than UV into the skin, with approximately 50% reaching the dermis [1–3]. The main source of IR radiation is the sun; the actual solar dose reaching the skin is influenced by several factors: ozone layer, position of the sun, latitude, altitude, cloud cover, and ground reflections. Based on these parameters, it should be noted that the overall composition of sunlight, e.g., in terms of the UV/IRA ratio is changing throughout the day. In addition to natural sunlight, artificial IR sources are constantly gaining importance; they are used
for therapeutic as well as for lifestyle purposes. While therapeutic use of IRA provides beneficial effects, for example, in wound healing, lifestyle-motivated applications of IRA, e.g., for ‘‘wellness’’ irradiations or for means of skin rejuvenation appear to be quite paradoxical [4].
Infrared Radiation and Skin Aging The role of IR radiation in premature skin aging was described over 20 years ago by L. Kligman [5]. She was the first to report that infrared radiation enhances UV-induced skin damage in guinea pigs. This prompted her to investigate the effect of IR alone; as a consequence, she could demonstrate that IR leads to elastosis, with ‘‘IR inducing the production of many fine, feathery fibers’’ and ‘‘a large increase in ground substance, a finding also seen in actinically damaged human skin.’’ From these observations, she has concluded that IR radiation contributes to skin aging. It took, however, almost 20 years until the underlying molecular mechanisms could be identified.
Molecular Mechanisms Schieke et al. reported in 2002 that low, physiologically relevant doses of IRA lead to a disturbance of the dermal extracellular matrix. IRA irradiation results in an induction of Matrixmetalloproteinase-1 (MMP-1) in vitro in human dermal fibroblasts, while expression of the respective tissue inhibitor TIMP-1 was not increased. This finding has, since then, been confirmed in independent studies by different workgroups in vitro and in vivo [6, 7]. Matrixmetalloproteinases (MMPs) are zinc-dependent endopeptidases responsible for the degradation of extracellular matrix components such as collagen and elastin. Under physiological conditions, MMPs are part of a coordinate network and are precisely regulated by their endogenous inhibitors, tissue inhibitors of MMPs (TIMPs). The unbalanced activity of MMPs with excessive proteolysis is thought to be a major pathophysiological factor in extrinsic skin aging. The increased expression of MMPs without a respective increase in TIMP expression results in the
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_42, # Springer-Verlag Berlin Heidelberg 2010
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Infrared A-induced Skin Aging
. Figure 42.1 Skin penetration of infrared radiation. Different wavelengths of natural and artificial radiations have different penetration capabilities. IRA penetrates well into the skin, approximately 50% of IRA is absorbed in the dermis. IRB reaches the dermis as well, while IRC is nearly completely absorbed in the epidermis
cleavage of fibrillar collagen, and thus impairs the structural integrity of the dermis [8–10]. This impairment can be partially countered by an increased expression of collagen itself. It is therefore important to note that IRA has recently been found to decrease the expression of the dominant human collagen gene Col1a1 in vitro and in vivo [11, 6]. Taken together, IRA disturbs the collagen equilibrium of the skin in two ways: (1) by increasing the amount/ activity of MMP-1, which results in an increased collagen degradation and (2) by decreasing de novo synthesis of collagen. While the biological endpoints of IRA irradiation resemble those found after UV irradiation, the underlying cellular molecular processes are completely different. This is particularly evident if UVA and IRA are being compared: the primal event in both cases is an increased amount of reactive oxygen species (ROS), which on a first glare seems to indicate a similarity rather than a difference. More detailed analysis – however – revealed huge differences between UVA and IRA. UVA induces an increased production of ROS by NADPH-oxidases, which are located in the cytoplasma membrane [12] and in addition repetitive
UVA irradiation results in damage to the mitochondrial DNA (mtDNA) [13]. IRA, on the other hand acts via a disturbance of the mitochondrial electron transport chain (mtETC). This multiprotein facility, driven by reduction equivalents (NADH/H + and FADH2), is responsible for energy conservation by transferring electrons to oxygen, while building up an electrochemical proton gradient across the inner mitochondrial membrane, which in turn fuels the production of ATP from ADP and Pi. As this process is not error free, relatively small amounts of ROS are always generated. Upon IRA irradiation this amount is significantly increased [4]. ROS are often recognized only as damaging agent, but they are well known to function in terms of cellular signaling. Reactive oxygen species (ROS) can serve to trigger molecular signaling responses and several studies indicate that ROS cause an inactivation of protein-tyrosine phosphatases (PTPs) by oxidizing conserved cysteine residues in the active sites of PTPs and thereby lead to a net increase in kinase phosphorylation/activation [14]. After IRA irradiation, not only the mitochondrial levels, but also the cellular ROS levels are increased and a disturbance of the cellular glutathione (GSH) equilibrium is observed [15]. GSH is one of the most important endogenous antioxidants; it can prevent or repair oxidative damage, and as a consequence it is oxidized itself, forming the glutathione dimer (GSSG). In this regard, IRA irradiation leads to a significant shift of the GSH/ GSSG equilibrium towards the oxidized form [15]. IRA-induced ROS production is not just a by-product of the irradiation, but of functional relevance because boosting the cellular antioxidative defense by increasing the cellular GSH content abrogated the IRA-induced upregulation of MMP-1 [15]. In addition, use of specific antioxidants in cell culture has also been shown to decrease the IRA-induced effects [7]. Mitochondria are known to act as a hub for cellular signaling with disruption of the mtETC being a prominent inducer of such retrograde (i.e., from mitochondria to nucleus) signaling [16]. In contrast to anterograde signaling processes here the nuclear gene expression is regulated by events originating in the mitochondria. The IRA-induced increase in mitochondrial ROS was recently found to initiate such a retrograde signaling cascade (> Fig. 42.2). Downstream of mitochondrial ROS, the IRA radiationinduced signaling pathway relevant for MMP-1 induction has been found to involve the activation of MAPKinases. Three distinct MAPK pathways have been characterized: the extracellular signalregulated kinase 1/2 (ERK1/2) pathway (Raf-MEK1/2-ERK1/2), the c-Jun N-terminal kinase
Infrared A-induced Skin Aging
. Figure 42.2 Infrared A-induced signal transduction. IRA radiation leads to an increased amount of mitochondrial ROS, which in turn leads to initiation of retrograde signaling, finally resulting in an increased expression of MMP-1 mRNA and protein and a decreased expression of Col1a1
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subsequent study by the same group indicates that IRA influences cutaneous wound repair by altering the levels of transforming growth factor (TGF)-b1 and MMP-2 [21]. Yet another study showed an influence of IRA on protein expression of ferritin: an increased ferritin expression was detected after IRA irradiation of keratinocytes and fibroblasts [22]. Ferritin is involved in the cellular antioxidative defense and the induction of this putative defense system in human skin most likely reflects a cellular response to oxidative processes triggered by IRA. Frank et al. showed that IRA interferes with apoptotic pathways, namely the mitochondrial apoptosis pathway [23] and reported that IRA signals via p53 [24]. The abrogating effect of IRA on apoptosis induced by lethal doses of extrinsic factor has recently been confirmed by another study [25].
Dosimetry of IRA
pathway (MEKK1/3-MKK4/7-JNK1/2/3), and p38 (MEKKMKK3/6-p38 a–d) pathway also termed stress-activated protein kinases (SAPKs). The ERK1/2 pathway is primarily induced by mitogens such as growth factors, whereas the SAPK pathways are predominantly induced by inflammatory cytokines as well as environmental stress such as UV, heat, and osmotic shock. Activated MAPKs translocate to the nucleus, where they phosphorylate and activate transcription factors such as c-Jun, c-Fos, ATF-2, and ternary complex factors (TCF) leading to the formation and activation of homo- or heterodimeric forms of the transcription factor AP-1. The promoter region of MMP-1 carries multiple AP-1-binding sites. For IRA, it has been demonstrated that ERK1/ 2 and p38 are activated in dermal fibroblasts, but that only inhibition of ERK1/2 activation subdues the IRAinduced increase of MMP-1 (reviewed in [17]). Although up to now the main research focus has been on MMP-1 and Col1a1 it is very likely that the IRAinduced activation of MAPKinases affects the regulation of other genes as well. Indeed, several additional effects of IRA are known: Kim et al. reported that infrared exposure is involved in neoangiogenesis in human skin, because IRA induces an angiogenic switch by altering the balance between the angiogenic inducer VEGF and the angiogenic inhibitor TSP-2 [18]. Interestingly, increased neoangiogenesis is a prominent feature of photoaged human skin [19]. Others found that IRA irradiation led to a decrease in epidermal proliferation, Langerhans cell density, and contact hypersensitivity reaction in mice [20], and a
Human dermal fibroblasts withstand IRA doses up to at least 1,200 J/cm2 [26], but the gene regulatory effects can already be observed at much lower, physiologically relevant dosage, i.e., 54 [8], 240 [4], or 360 J/cm2 [15]. Increased levels of cytosolic and mitochondrial ROS were detected even after a treatment with 30 J/cm2 [15].
IRA Chromophores While the endogenous chromophores for IR are very likely to be part of the mtETC [27] and remain to be identified, several exogenous chromophores for IR are known. They are used for therapeutic purposes, e.g., in photodynamic therapy, and include palladium-bacteriopheophorbide and indocyanine green [28, 29].
Protection Against IRA Up to now, photoprotection of human skin has focused against UVB and/or UVA radiation. The studies discussed above indicate, however, that protection against IRA radiation has to be included in order to achieve complete protection. In this regard antioxidants appear to be promising. Based on the fact that mtROS are functionally relevant in the IRA-induced effects, antioxidants that target the mitochondria theoretically represent potential IRA protective substances. Indeed, it has been demonstrated in vitro and in vivo that such specific antioxidants protect against detrimental IRA effects, e.g., IRA-induced MMP-1 expression [7].
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In contrast, there are currently no chemical or physical UV filters available, which are suited for commercial suncare products, and which have been shown to provide IRA protection. The protective effect of textiles remains to be evaluated in terms of IRA protection. There is, however, data available showing that use of a black cloth at least partially provides IRA protection [18]. Finally, the topic of avoidance has to be discussed. Until now, there is no information source available that would provide a measure on the actual IRA load that would be comparable to the well-established UV index. Establishing a respective IRA index might be a considerable contribution.
10.
Conclusion
13.
As skin aging is a complex process, it is not surprising that ongoing research efforts uncover more and more environmental factors enfolding detrimental effects on the skin. Regarding natural sunlight or artificial sources of its components there is a major doubt that whether in addition to UV, IRA protection also has to be taken into account. IRA photoprotection requires specialized strategies with topical application of mitochondrially targeted antioxidants being a promising option.
References 1. Kochevar IE, Taylor CR, Krutmann J. Fundamentals of cutaneous photobiology and photoimmunology. In: Wolff K, Austen KF, Goldsmith LA, Katz SI, Gilchrest BA, Paller AS, Leffel DJ (eds) Fitzpatrick’s Dermatology in General Medicine. New York: McGraw-Hill, 2007. 2. Cobarg CC. Physikalische Grundlagen der wassergefilterten InfrarotA-Strahlung. In: Vaupel P, Kru¨ger W (eds) Wa¨rmetherapie mit wassergefilterter Infrarot-A-Strahlung. Stuttgart: Hippokrates Verlag, 1995, pp. 19–28. 3. Hellige G, Becker G, Hahn G. Temperaturverteilung und Eindringtiefe wassergefilterter Infrarot-A-Strahlung. In: Vaupel P, Becker G (eds) Wa¨rmetherapie mit wassergefilterter Infrarot-A-Strahlung. Stuttgart: Hippokrates Verlag, 1995, pp. 63–80. 4. Schroeder P, Haendeler J, Krutmann J. The role of near infrared radiation in photoaging of the skin. Exp Gerontol. 2008; 43:629–632. 5. Kligman LH. Intensification of ultraviolet-induced dermal damage by infrared radiation. Arch Dermatol Res. 1982;272:229–238. 6. Kim MS, Kim YK, Cho KH, Chung JH. Regulation of type I procollagen and MMP-1 expression after single or repeated exposure to infrared radiation in human skin. Mech Ageing Dev. 2006;127:875–882. 7. Schroeder P, Lademann J, Darvin ME, Stege H, Marks C, Bruhnke S, Krutmann J. Infrared radiation-induced matrix metalloproteinase in
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human skin: implications for protection. J Invest Dermatol. 2008;128:2491–2497. Brenneisen P, Sies H, Scharffetter-Kochanek K. Ultraviolet-B irradiation and matrix metalloproteinases: from induction via signaling to initial events. Ann N Y Acad Sci. 2002;973:31–43. Fisher GJ, Kang S, Varani J, Bata-Csorgo Z, Wan Y, Datta S, Voorhees JJ. Mechanisms of photoaging and chronological skin aging. Arch Dermatol. 2002;138:1462–1470. Fisher GJ, Wang ZQ, Datta SC, Varani J, Kang S, Voorhees JJ. Pathophysiology of premature skin aging induced by ultraviolet light. N Engl J Med. 1997;337:1419–1428. Buechner N, Schroeder P, Kunze K, Maresch T, Calles C, Krutmann J, Haendeler J. Thioredoxin-1 protects from MMP-1 upregulation and collagen type Ia1 downregulation: implication for photoaging. Exp Gerontol. 2008 (this issue). Schauen M, Hornig-Do HT, Schomberg S, Herrmann G, Wiesner RJ. Mitochondrial electron transport chain activity is not involved in ultraviolet A (UVA)-induced cell death. Free Radic Biol Med. 2007;42:499–509. Berneburg M, Plettenberg H, Medve-Konig K, Pfahlberg A, Gers-Barlag H, Gefeller O, Krutmann J. Induction of the photoaging-associated mitochondrial common deletion in vivo in normal human skin. J Invest Dermatol. 2004;122:1277–1283. Cross JV, Templeton DJ. Regulation of signal transduction through protein cysteine oxidation. Antioxid Redox Signal. 2006;8:1819–1827. Schroeder P, Pohl C, Calles C, Marks C, Wild S, Krutmann J. Cellular response to infrared radiation involves retrograde mitochondrial signaling. Free Radic Biol Med. 2007;43:128–135. Butow RA, Avadhani NG. Mitochondrial signaling: the retrograde response. Mol Cell. 2004;14:1–15. Schieke SM, Schroeder P, Krutmann J. Cutaneous effects of infrared radiation: from clinical observations to molecular response mechanisms. Photodermatol Photoimmunol Photomed. 2003;19:228–234. Kim MS, Kim YK, Cho KH, Chung JH. Infrared exposure induces an angiogenic switch in human skin that is partially mediated by heat. Br J Dermatol. 2006;155:1131–1138. Yaar M. Clinical and histological features of intrinsic versus extrinsic skin aging. In: Gilchrest BA, Krutmann J (eds) Skin Aging. New York: Springer, 2006, pp. 9–21. Danno K, Sugie N. Effects of near-infrared radiation on the epidermal proliferation and cutaneous immune function in mice. Photodermatol Photoimmunol Photomed. 1996;12:233–236. Danno K, Mori N, Toda K, Kobayashi T, Utani A. Near-infrared irradiation stimulates cutaneous wound repair: laboratory experiments on possible mechanisms. Photodermatol Photoimmunol Photomed. 2001;17:261–265. Applegate LA, Scaletta C, Panizzon R, Frenk E, Hohlfeld P, Schwarzkopf S. Induction of the putative protective protein ferritin by infrared radiation: implications in skin repair. Int J Mol Med. 2000;5:247–251. Frank S, Oliver L, Lebreton-De Coster C, Moreau C, Lecabellec MT, Michel L, Vallette FM, Dubertret L, Coulomb B. Infrared radiation affects the mitochondrial pathway of apoptosis in human fibroblasts. J Invest Dermatol. 2004;123:823–831. Frank S, Menezes S, Lebreton-De Coster C, Oster M, Dubertret L, Coulomb B. Infrared radiation induces the p53 signaling pathway: role in infrared prevention of ultraviolet B toxicity. Exp Dermatol. 2006;15:130–137. Jantschitsch C, Majewski S, Maeda A, Schwarz T, Schwarz A. Infrared radiation confers resistance to UV-induced apoptosis
Infrared A-induced Skin Aging via reduction of DNA damage and upregulation of antiapoptotic proteins. J Invest Dermatol. 2009;129(5):1271–1279, Epub 2008 Nov 27. 26. Schieke S, Stege H, Kurten V, Grether-Beck S, Sies H, Krutmann J. Infrared-A radiation-induced matrix metalloproteinase 1 expression is mediated through extracellular signal-regulated kinase 1/2 activation in human dermal fibroblasts. J Invest Dermatol. 2002;119:1323–1329. 27. Karu T. Primary and secondary mechanisms of action of visible to near-IR radiation on cells. J Photochem Photobiol B. 1999;49:1–17.
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28. Koudinova NV, Pinthus JH, Brandis A, Brenner O, Bendel P, Ramon J, Eshhar Z, Scherz A, Salomon Y. Photodynamic therapy with Pd-Bacteriopheophorbide (TOOKAD): successful in vivo treatment of human prostatic small cell carcinoma xenografts. Int J Cancer. 2003;104:782–789. 29. Tseng WW, Saxton RE, Deganutti A, Liu CD. Infrared laser activation of indocyanine green inhibits growth in human pancreatic cancer. Pancreas. 2003;27:e42–e45.
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15 Neurotrophins and Skin Aging Mohamed A. Adly . Hanan Assaf . Mahmoud R. Hussein
Introduction Cutaneous aging is a complex biological phenomenon that consists of two superimposed components: intrinsic (true aging) and extrinsic (photoaging) aging. Intrinsic aging is largely genetically determined and represents an inevitable change attributable to the passage of time alone. It resembles aging that is seen in most internal organs and its underlying mechanisms probably involve decreased proliferative capacity, leading to cellular senescence and altered biosynthetic activity of skin-derived cells. Intrinsic aging is manifested primarily by physiologic alterations with subtle but undoubtedly important consequences for both healthy and diseased skin. The morphologic changes of intrinsic aging include smoothing and thinning of the skin with exaggeration of the expression lines. The intrinsic rate of skin aging in any individual is dramatically influenced by personal and environmental factors, particularly the amount of exposure to ultraviolet light (UV), that is, intrinsic and extrinsic aging are superimposed processes. Extrinsic aging is caused by environmental exposure, primarily to UV. It is observed in the sun-exposed areas (photoaging) and is manifested by the presence of skin wrinkles, pigmented lesions, patchy hypopigmentations, and actinic keratoses. It involves changes in the cellular biosynthetic activity and usually leads to gross disorganization of the dermal matrix. Photodamage, which considerably accelerates the visible aging of skin, also greatly increases the risk of cutaneous neoplasia. The molecular mechanisms underlying skin aging are poorly understood. They seem to be a multifaceted process influenced by various factors affecting different body sites at variable degrees. This chapter discusses the possible roles of some molecules involved in cutaneous aging, namely neurotrophins (NTs).
Overview of Neurotrophins Neurotrophins (NTs) are a family of structurally and functionally related polypeptides, which show about 50% amino acid sequence homology. NTs belong to a
family of growth factors, which control the development, maintenance, and apoptotic death of neurons. They also have multiple regulatory functions outside the peripheral and central nervous systems [1–3]. The NTs family consists of four structurally and functionally related proteins known as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). It is well known that all four members of the NTs family are synthesized as precursors which are cleaved by intracellular proteases to release the C-terminal mature proteins [4]. Mature NTs proteins are approximately 13 kDa in size and share about 50% of amino acid sequence homology. They exert their biological effects as dimers interacting with specific receptors. High-affinity receptors for NTs belong to the tyrosine kinase family. Tyrosine kinase receptor A (TrkA) is the highaffinity receptor for NGF, tyrosine kinase receptor B (TrkB) is the high-affinity receptor for BDNF and NT-4, and tyrosine kinase receptor C (TrkC) is the high-affinity receptor for NT-3 [5]. However, NT-3 may also bind to TrkA and TrkB receptors, but with low affinity. All four NTs interact with the low-affinity p75 kDa NT receptor (p75NTR), which is a member of the tumor necrosis factor family of receptors containing the cytoplasmic ‘‘death’’ domain, involved in mediating a number of responses independently or in association with Trk receptors [4, 6]. By interacting with Trk receptors and/or p75NTR, NTs induce a variety of biological responses in neurons as well as in non-neuronal cells. They control proliferation, differentiation, and survival, whereby these interactions occur. The signals promoting survival or differentiation are generated by NT interaction with Trk receptors and require receptor dimerization, autophosphorylation, and the subsequent involvement of a number of adaptor molecules coupling Trk receptors to the distinct intracellular signal transduction pathways [5]. NTs modulate synaptic transmission via Trk-associated regulation of intracellular Ca2+, promote survival via phosphorylation and inactivation of several proapoptotic substrates including Bad. They promote differentiation via activation of the Ras/Raf/ERK kinase/mitogen-activated protein kinase cascade [5]. p75NTR performs distinct functions depending on whether it is coexpressed with Trk receptors and/or
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_15, # Springer-Verlag Berlin Heidelberg 2010
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selected other growth factor receptors (sortilin, Nogo receptor complex), or whether it is expressed alone. The coexpression of p75NTR with Trk receptors increases high-affinity NT binding, enhances Trk ability to discriminate a preferred ligand from the other NTs, and promotes survival effects of the NTs [4, 6]. When p75NTR is coexpressed with sortilin (a non-G-protein-coupled neurotensin receptor), NT precursor proteins (pro-NTs) interacting with p75NTR–sortilin complex induce apoptotic death [7]. In case of coexpression of p75NTR with the Nogo receptor complex, Nogo induces growth inhibition [4]. When p75NTR is expressed alone on the cell surface, mature NT peptides or selected non-NT ligands (beta-amyloid or a fragment of the prion protein) are capable of inducing apoptosis or promote survival depending on the intracellular adaptor molecules present in target cells [4, 8]. Apoptotic signaling via p75NTR requires the presence of intracellular adaptor molecules (NT receptor-interacting factors 1 and 2, NT receptorinteracting MAGE homolog, and NT-associated death executor) that link p75NTR signaling with the JNK-p53Bax proapoptotic pathway. However, signaling through p75NTR expressed alone – besides inducing apoptosis – may also promote cell survival. Intracellular adaptor molecules interacting with the C-terminus of p75NTR (TNF receptor-associated factor 6, Fas-associated phosphatase-1, and receptor-interacting protein-2) link p75NTR with the NF-kb pathway and can thus promote survival. However, mechanisms involved in controlling the expression and preferential engagement of adaptor molecules in distinct cell types remain to be clarified.
NT Family Members are Expressed in the Mammalian Skin NTs were originally discovered in the nervous system where they were found to be involved in the differentiation and survival of the neurons. However, they were found to be expressed in a variety of tissues outside the nervous system where they have non-neuronal targets in the skin [3, 9–11], kidney, tooth, muscle, and heart [12]. In mice, NTs are expressed very early during embryonic development (E9.5–E10.5) in both the skin epithelium and the cutaneous mesenchyme [13]. The onset of NT expression in embryonic skin coincides with the time point of appearance of K5 and K14 in the epidermis (E9.5), whereas maximal NT synthesis coincides with the beginning of vibrissa development in facial skin (E12.5), and with the initiation of tylotrich hair follicle (HF) induction in dorsal murine skin (E14.5) [13]. This raises
the hypothesis that NTs fulfill multiple non-neurotrophic functions during skin development. In murine postnatal skin, NTs and their receptors are differentially distributed in distinct cell populations. NGF and NT-3 are expressed by basal epidermal keratinocytes in mice and humans [12, 14–21] (> Fig. 15.1). They are also produced by fibroblasts in vitro, and NGF stimulates fibroblast migration [14, 15]. In situ, BDNF and NT-3 are expressed in cutaneous nerve fibers and myocytes of the arrector pili and panniculus carnosus muscles of mice [16, 17]. In adult human skin, NGF and NT-3 are also expressed by fibroblasts, arrector pili muscle, sebaceous and sweat glands, and hair follicles [15, 16] (> Figs. 15.2 and > 15.3). NT receptors (TrkA, TrkC, and p75NTR) have been detected on human epidermal keratinocytes [12, 14–19]. In murine skin, only TrkA and TrkB isoforms are seen in epidermal keratinocytes, whereas TrkC and p75NTR are expressed in cutaneous nerves and in the HF [16, 17].
NTs and Epidermal Homeostasis Work over the past 10 years has indicated that NTs possess a range of functions outside the nervous system [12, 18, 19] and can be considered as growth factors in epithelial tissue homeostasis. It was demonstrated that normal human keratinocytes synthesize and secrete biologically active NGF [14, 20, 21]. In human skin, NGF is released in increasing amounts by proliferating keratinocytes, whereas secretion ends in more differentiated cells [22, 23]. Both exogenous and endogenous NGF are capable of inducing keratinocyte proliferation [12, 14–19]. On the other hand, in the presence of their normal mesenchymal environment, exogenous NGF can indeed either stimulate or inhibit murine epidermal and HF keratinocyte proliferation in situ, depending on whether the keratinocytes are in a state of relative quiescence or are already maximally proliferating [24]. The proliferative effects of autocrine NGF on human keratinocytes are also confirmed by the use of the natural alkaloid K252a, an inhibitor of TrkA phosphorylation. Indeed, K252 blocks keratinocyte proliferation, in the absence of exogenous NGF [18]. Moreover, human keratinocytes transfected with NGF proliferate to a significantly greater extent than mocktransfected cells [25]. Keratinocytes express and release NTs, other than NGF [16, 17, 26], and BDNF, NT-3, and NT-4 stimulate murine epidermal keratinocyte proliferation in situ [16, 17]. NGF is secreted at highest levels as compared to the other NTs, whereas NT-3 and NGF upregulate each other’s secretion in human keratinocytes. NGF expression
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. Figure 15.1 Expression of NTs and their receptors in normal human skin shown in red color with tyramide signal amplification (TSA) and avidin–biotin complex (ABC) immunostaining techniques. (A) Nerve growth factor (NGF) (200¥ ), (B) NT3 (200¥ ), (C) Tyrosine Kinase A (TrkA) (200¥ ), (D) TrkC (200¥ ), (E): p75NTR (200¥ ), F, G, H, I and J refer to as the expressions of NGF, NT-3, TrK A, TrK C and p75NTR, respectively, confirmed with ABC immunostaining. (INF) Infundibulum. Adapted from Adly et al. [17, 21] ß 2005, 2006, Wiley Blackwell and Reprinted with permission from Adly et al. [32] ß 2009, Elsevier
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. Figure 15.2 Immunoreactivity of nerve growth factor (NGF) protein in adult human scalp anagen VI HF (hair Follicle), shown in red color with avidin–biotin complex (ABC) and tyramide signal amplification (TSA) techniques. A, B, F, and G show IR in the distal region of HF. C, D, and H show immunoreactivity (IR) in the mid region of hair follicle HF. E and I show IR in the proximal bulb region of HF. J is a schematic summary representation of IR in the whole HF shown in red color. K shows IR in catagen HF. l shows IR in telogen HF. CTS connective tissue sheath; DP dermal papilla; HCo hair cortex; HMe hair medulla; HMC hair matrix cells; HS hair shaft; INF infundibulum; IRS inner root sheath; ORS outer roof sheath. (Reprinted with permission from Adly et al. [21]) ß 2006, Wiley-Blackwell
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. Figure 15.3 Immunoreactivity of NT-3 in human scalp skin and HF, shown in red color with avidin–biotin complex (ABC) and tyramide signal amplification (TSA) techniques. A and G show the epidermis. B–F (panel 1) and H–L (panel 2) show IR in anagen VI HF. C and J show IR in the sebaceous gland (SG). N and R show IR in the sweat gland. O shows IR in the early anagen, some fibroblasts in the dermis, and adipocytes in subcutis. M and P show IR in telogen HF. Q shows IR in catagen HF. S is a schematic summary representation of IR in anagen VI HF shown in red color. APM arrector pili muscle. (Reprinted with permission from Adly et al. [17]) ß 2005, Wiley-Blackwell
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is downregulated by UVB irradiation [23, 25], whereas NT-3 release is augmented by UVA. At the skin level, TrkA and TrkC mediate NGF- and NT-3-induced keratinocyte proliferation, respectively. Indeed, keratinocytes overexpressing TrkA proliferate significantly better than controls [27], and increasing concentrations of anti-NT-3 antibody inhibit keratinocyte proliferation [26]. In mouse skin, epidermal keratinocytes express TrkA and TrkB, and all NTs are capable of stimulating their proliferation in ex vivo-cultured skin explants [16, 17, 24]. In human skin, epidermal keratinocytes express TrkA, TrkB, and TrkC proteins in different intensities [21] and NTs can stimulate keratinocyte proliferation and epidermal homeostasis. Apoptosis plays a fundamental role in epidermal homeostasis by counterbalancing cell proliferation, and apoptotic cells are consistently present in normal human epidermis [28–30]. In vitro, NGF, but not the other NTs, can rescue human epidermal keratinocytes from spontaneous and UVB-induced apoptosis via TrkA [25, 28, 29]. Although UVB downregulates NGF and TrkA in human keratinocytes, NGF-overexpressing keratinocytes are protected from UVB-induced apoptosis [25]. NGF protects keratinocytes from cell death via the Bcl-2 family of apoptosis inhibitors. Indeed, K252a fails to induce apoptosis in keratinocytes overexpressing Bcl-2, and UVB causes a decrease in Bcl-2 and Bcl-xL expression in mock-transfected keratinocytes, but not in NGFoverexpressing cells. NGF prevents the cleavage of the enzyme poly (ADP-ribose) polymerase, a substrate for caspases, which is induced in human keratinocytes by UVB [25]. These observations are consistent with a model whereby autocrine NGF protects human keratinocytes from apoptosis through its high-affinity receptor TrkA by maintaining constant levels of Bcl-2 and Bcl-xL, which in turn block caspase activation [25]. The above-mentioned observations clearly show that NTs mediate proliferative and survival signals in epidermal keratinocytes through their high-affinity Trk receptors. Still, the role of the low-affinity p75NTR in NGF signaling in keratinocytes remains to be clarified. Although TrkA is evenly distributed in the basal keratinocyte layer (> Fig. 15.1c), p75NTR is expressed in basal keratinocytes with an irregular pattern (> Fig. 15.1e). As human keratinocytes lack functional TrkB [25], BDNF and NT-4 obviously signal through p75NTR in these cells. Indeed, BDNF and NT-4 induce apoptosis in cultured human keratinocytes. This is in agreement with the observation of a similar function of p75NTR in the catagen phase of the hair cycle [31] and the strong
expression of p75NTR protein in the anagen–catagen transition and early catagen stages of the human hair follicle cycle [32]. Therefore, a balance between the lowand the high-affinity NT receptors exists in keratinocytes. However, the exact stimuli and conditions whereby NGF and other NTs signal life or death in keratinocytes are yet to be defined. In addition, it should also be determined whether NTs and their receptors could play a role in the development of non-melanoma skin cancers by stimulating proliferation and inhibiting apoptosis, in a manner similar to what has been shown for prostate [33] and breast neoplasia [34].
NTs and Melanocytes During skin development, neural crest-derived melanoblasts migrate into the skin and differentiate into melanocytes, which populate the basal layer of the epidermis and the HFs. Together with other paracrine signaling molecules (fibroblast growth factor [FGF], bone morphogenetic proteins, noelin-1, stem cell factor, hepatocyte growth factor, endothelins), NTs play an important role in the control of melanoblast migration, viability, and differentiation [14, 29]. Normal human melanocytes also express p75NTR [32] and its expression level is upregulated by a variety of stimuli including UV irradiation [35]. Keratinocyte-derived NGF, whose expression is also upregulated by UV irradiation [20, 36], may influence epidermal melanocytes in a paracrine manner. In vitro, NGF is chemotactic for melanocytes and stimulates melanocyte dendrite formation [14]. Although under optimal basal culture conditions, there is no effect of NGF on melanocyte cell yields or melanogenesis, both NGF and NT-3, the latter expressed by dermal fibroblasts [15], increase melanocyte survival when the cells are maintained in medium depleted of growth factors [15, 37]. Interestingly, phorbol 12-tetra decanoate 13 acetate, a strong activator of protein kinase C, upregulates the expression of p75NTR and induces the expression of TrkA in melanocytes [15]. Although the exact mechanism that regulates phorbol 12-tetra decanoate 13 acetate-induced p75NTR and TrkA upregulation is not known, phorbol 12-tetra decanoate 13 acetate is recognized to have a striking effect also on melanocyte dendricity. It is possible that this differentiated morphology of melanocytes is part of an integrated complex of differentiated functions that includes induction of receptors to NGF. In contrast with TrkA expression that requires induction, melanocytes constitutively express TrkC, albeit the
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expression is likely to be low as it was detected by the sensitive reverse transcriptase-PCR methodology [15]. Also, in contrast with TrkA expression, TrkC expression is decreased after phorbol 12-tetra decanoate 13 acetate, suggesting that although melanocytes can bind both NGF and NT-3, different signals that preferentially induce a specific high-affinity receptor determine which NT would exert its effect. Thus, the effects of NGF and NT-3 on melanocytes may be influenced by outside signals through modulations of their high-affinity receptor expression. Indeed, using UV-irradiated cultured melanocytes and human melanoma cells, NGF supplementation enhances cell survival, markedly reduces apoptotic cell death, and increases the level of the antiapoptotic Bcl-2 protein, which is expressed strongly by melanocytes in vivo even in the absence of UV irradiation [23, 37]. The data suggest that NGF, which is constitutively produced by neighboring epidermal keratinocytes, may preserve the population of cutaneous melanocytes that would otherwise be depleted by sun exposure. In contrast, NT-3, which is strongly expressed by nonproliferating fibroblasts [15], like those in the dermal compartment of nondamaged human skin, could help in melanocyte maintenance during steady state conditions.
NTs Expression in Human Skin Decreases with Aging Recent studies have revealed that the expression of NTs and Trk receptors within the human skin decreases with aging. NGF is a member of a family of structurally and functionally related polypeptides known as the neurotrophins (NTs) [38]. Since its discovery, NGF is known to guide and sustain neuronal development and differentiation within peripheral neural networks. NGF can regulate tissue morphogenesis, remodeling, proliferation, and apoptosis [3]. The common neuroectodermal origin of the cutaneous epithelium and the nervous system makes it reasonable to hypothesize that the same growth factors, which govern the development, and maintenance, of neurons are also involved in skin morphogenesis [3]. The NGF is established locally in the skin by glia cells, epithelial cells, fibroblasts and Merkel cells. The skin is a rich source of NGF and the epidermis is recognized as a site of NGF expression. Shortly thereafter, it became clear that epidermal keratinocytes are not only important NGF sources, but also are NT targets expressing NT receptors. In murine skin organ cultures, NGF is produced by keratinocytes. Also, NGF
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stimulates the proliferation and inhibits the apoptosis in cultured human epidermal keratinocytes. Interestingly, NGF is critical for proper innervation of this peripheral sensory organ. Thus, defects in NTs singling are associated with severe sensory skin disorders that inhibit wound healing [3].
Downregulation of NGF Protein Expression with Aging The expression pattern of NGF was examined previously in different age groups. NGF protein expression exhibited striking age-associated changes within the human epidermis [3]. In sun-protected skin specimens derived from young individuals (<40 years), NGF expression was very strong and was prominent in the most epidermal layers, particularly in the stratum basale, stratum spinosum, and stratum granulosum (> Fig. 15.4). In contrast, in the skin derived from old donors (>60 years), no or only a weak NGF expression was detected (> Fig. 15.4). Semiquantitative analysis of NGF immunostaining revealed that NGF protein expression values were significantly higher in young ages than in old ages (> Tables 15.1 and > 15.2). NGF immunoreactivity was strongest in the ages 6, 15, and 18 years and decreased gradually in the ages 33 and 39 years (> Fig. 15.4). In the young ages, the expression was detected in all layers of the epidermis except for the stratum corneum (> Fig. 15.4a–c), whereas in the middle ages (33 and 39 years) the expression was confined to the Malpighian layer and stratum spinosum (> Fig. 15.4d, e). In the old ages (>60 years), NGF expression was dramatically reduced and the expression was detected mainly in the stratum basale (> Fig. 15.4f–j). Among old ages (60–81 years), the expression was strongest in the age of 60 years (> Fig. 15.4f ), whereas it was greatly diminished in older ages (> Fig. 15.4g–i) until it became completely negative in the age of 81 years (> Fig. 15.4j). Although NGF expression was seen in the stratum granulosum of the skin derived from certain old ages (68- and 78-year-old donors), its immunoreactivity was weak (> Fig. 15.4g, i). The level of NGF protein in the sweat glands does not apparently differ with aging (> Fig. 15.5). The age-related decrease of NGF protein expression in the human epidermis is parallel with its level in the nervous system. There was a gradual reduction of NGF levels with aging in the brain and thymus of rats; and a low amount of NGF protein in the plasma of old subjects. Also, the administration of NGF in rats can reduce agerelated atrophy of the neurons. Similarly in human,
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. Figure 15.4 Nerve growth factor protein expression in human skin derived from different aged donors shown in red color with tyramide signal amplification (TSA) technique. (A–E) Young age group (5–39 years). (A) 6Y, (B) 15Y, (C) 18Y, (D) 33Y, and (E) 39Y. (F and G) Old age group (60–81 years). (F) 60Y, (G) 68Y, (H) 71Y, (I) 78Y, and (J) 81Y. Magnification: 200¥ (Reprinted with permission from Adly et al. [3] ) ß 2006, Elsevier
administration of NGF can delay retinal degeneration in patients with retinitis pigmentosa [39, 40]. The strong expression of NGF protein in the skin of young individuals may be due to an increased receptormediated internalization of NGF proteins released by nerve endings [41, 42], or altered expression of certain cytokines, such as tumor necrosis factor alpha (TNF-a), that influence the synthesis of NGF. In transgenic mice, the basal level of brain NGF can be influenced negatively or positively by local expression of TNF-a [19, 43]. The decrease of NGF protein expression in aged skin may reflect impairment of these mechanisms or a reduction in the number of high-affinity NGF binding sites. Indeed,
as women age, they become hypo-estrogenic; therefore, a hypothesis to be tested is that NGF changes with aging might by related to hormonal change, and not as much with aging. To test this hypothesis, NGF protein expression in skin of male subjects need to be examined in this type of studies.
Downregulation of NT-3 Protein Expression with Aging Similarly, NT-3 protein expression in human skin underwent age-associated decrease (Adly MA, 2009). In young
15
Neurotrophins and Skin Aging
. Table 15.1 Nerve growth factor protein expression in human skin of different ages. The staining results were examined by the authors and were scored as (–) for absent, (+) for weak, (++) for medium, and (+++) for intense nerve growth factor (NGF) protein expression Age (years)
Stratum basale
Stratum spinosum
Stratum granulosum
Stratum corneum
6
+++
+++
+++
–
8
+++
+++
+++
–
11
+++
+++
+++
–
13
+++
+++
+++
–
15
+++
+++
+++
–
18
+++
+++
+++
–
32
+++
++
++
–
33
+++
++
++
– –
34
+++
++
++
36
+++
++
++
–
37
+++
++
++
–
39
+++
++
++
–
60
++
+
–
–
64
+
+
–
–
68
+
–
–
–
71
+
–
–
–
74
+
–
–
–
76
+
–
–
–
78
+
–
–
–
81
+
–
–
–
. Table 15.2 Expression values of nerve growth factor in human skin of different ages. The immunoreactivity score (IR score) was evaluated by multiplying the percentage of positive cells (PP%) and the staining intensity (SI). First, the PP% was scored as 0 for <5%, 1 for 5–25%, 2 for 25–50%, 3 for 50–75%, and 4 for >75%. Second, the SI was scored as 1 for weak, 2 for medium, and 3 for intense staining, following other groups. Values between brackets represent the standard errors of mean Basal layer Age groups (years)
SI
Spinous layer
Granular layer
PP
IRS
SI
PP
IRS
SI
PP
IRS
6–18
2.8 {0.2}
3.8 {0.2}
11.0 {1.0}
2.8 {0.1}
3.8 {0.2}
11.0 {1.0}
2.8 {0.2}
3.7 {0.2}
10.5 {1.0}
19–50
2.8 {0.1}
3.7 {0.3}
6.8 {1.1}
1.8 {0.1}
3.1 {0.1}
5.7 {0.3}
1.7 {0.3}
2.6 {0.4}
5.1 {0.8}
51–81
1.0 {0.0}
1.8 {0.1}
1.8 {0.1}
1.1 {0.1}
1.4 {0.4}
1.4 {0.4}
0.75 {0.1}
0.70 {0.1}
0.9 {0.1}
ages, NT-3 expression was very strong and immunoreactivity was detected in almost all layers of the epidermis, including the stratum corneum in the ages below 20 years. Alternatively, in old ages, the NT-3 expression was very weak or completely absent.
Conclusion The neurotrophins are a family of polypeptide growth factors. These proteins are critical not only for the development, but also for the maintenance of the vertebrate
155
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Neurotrophins and Skin Aging
. Figure 15.5 Nerve growth factor protein expression in sweat glands derived from different aged donors. The reactivity appeared as red color (tyramide signal amplification [TSA] technique). Nerve growth factor (NGF) expression in the following ages in years (Y). (a) 6Y, (b)33Y, (c) 39Y, (d) 71Y, (e) 78Y, and (f) 81Y(Reprinted with permission from Adly et al. [3] ) ß 2006, Elsevier
nervous system. Recently, several leads indicate that these factors could have a broader role than their name might suggest, in particular, the putative role of NGF, and its receptor TrkA and NT-3 in cutaneous homeostasis and in skin aging. To date knowledge about the expression pattern of neurotrophins in skin
remains rudimentary. The chapter discussed the expression of neurotrophins and their receptors in different cutaneous structures based on the data obtained from the studies of the human scalp skin. The clinical and therapeutic ramifications of these studies are open for further investigations.
Neurotrophins and Skin Aging
References 1. Botchkareva NV, Botchkarev VA, Welker P, Airaksinen M, Roth W, Suvanto P, Muller-Rover S, Hadshiew IM, Peters C, Paus R. New roles for glial cell line-derived neurotrophic factor and neurturin: involvement in hair cycle control. Am J Pathol. 2000;156(3): 1041–1053. 2. Botchkarev VA, Botchkareva NV, Peters EM, Paus R. Epithelial growth control by neurotrophins: leads and lessons from the hair follicle. Prog Brain Res. 2004;146:493–513. 3. Adly MA, Assaf H, Hussein MR. Age-associated decrease of the nerve growth factor protein expression in the human skin: preliminary findings. J Dermatol Sci. 2006;42(3):268–271. 4. Teng KK, Hempstead BL. Neurotrophins and their receptors: signaling trios in complex biological systems. Cell Mol Life Sci. 2004; 61(1):35–48. 5. Segal RA. Selectivity in neurotrophin signaling: theme and variations. Annu Rev Neurosci. 2003;26:299–330. 6. Dechant G, Barde YA. The neurotrophin receptor p75(NTR): novel functions and implications for diseases of the nervous system. Nat Neurosci. 2002;5(11):1131–1136. 7. Nykjaer A, Willnow TE, Petersen CM. p75NTR–live or let die. Curr Opin Neurobiol. 2005;15(1):49–57. 8. Yaar M, Zhai S, Fine RE, Eisenhauer PB, Arble BL, Stewart KB, Gilchrest BA. Amyloid beta binds trimers as well as monomers of the 75-kDa neurotrophin receptor and activates receptor signaling. J Biol Chem. 2002;277(10):7720–7725. 9. Peters EM, Hansen MG, Overall RW, Nakamura M, Pertile P, Klapp BF, Arck PC, Paus R. Control of human hair growth by neurotrophins: brain-derived neurotrophic factor inhibits hair shaft elongation, induces catagen, and stimulates follicular transforming growth factor beta2 expression. J Invest Dermatol. 2005;124 (4):675–685. 10. Peters EM, Hendrix S, Golz G, Klapp BF, Arck PC, Paus R. Nerve growth factor and its precursor differentially regulate hair cycle progression in mice. J Histochem Cytochem. 2006;54 (3):275–288. 11. Botchkareva NV, Botchkarev VA, Albers KM, Metz M, Paus R. Distinct roles for nerve growth factor and brain-derived neurotrophic factor in controlling the rate of hair follicle morphogenesis. J Invest Dermatol. 2000;114(2):314–320. 12. Sariola H. The neurotrophic factors in non-neuronal tissues. Cell Mol Life Sci. 2001;58(8):1061–1066. 13. Ernfors P, Lee KF, Jaenisch R. Target derived and putative local actions of neurotrophins in the peripheral nervous system. Prog Brain Res. 1994;103:43–54. 14. Yaar M, Grossman K, Eller M, Gilchrest BA. Evidence for nerve growth factor-mediated paracrine effects in human epidermis. J Cell Biol. 1991;115(3):821–828. 15. Yaar M, Eller MS, DiBenedetto P, Reenstra WR, Zhai S, McQuaid T, Archambault M, Gilchrest BA. The trk family of receptors mediates nerve growth factor and neurotrophin-3 effects in melanocytes. J Clin Invest. 1994;94(4):1550–1562. 16. Botchkarev VA, Metz M, Botchkareva NV, Welker P, Lommatzsch M, Renz H, Paus R. Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 act as ‘‘epitheliotrophins’’ in murine skin. Lab Invest. 1999;79(5):557–572. 17. Adly MA, Assaf HA, Nada EA, Soliman M, Hussein M, Human scalp skin and hair follicles express neurotrophin-3 and its high-affinity
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receptor tyrosine kinase C, and show hair cycle-dependent alterations in expression. Br J Dermatol. 2005;153(3):514–520. Bonini S, Rasi G, Bracci-Laudiero ML, Procoli A, Aloe L. Nerve growth factor: neurotrophin or cytokine? Int Arch Allergy Immunol. 2003;131(2):80–84. Aloe L. Nerve growth factor, human skin ulcers and vascularization. Our experience. Prog Brain Res. 2004;146:515–522. Di Marco E, Marchisio PC, Bondanza S, Franzi AT, Cancedda R, De Luca M. Growth-regulated synthesis and secretion of biologically active nerve growth factor by human keratinocytes. J Biol Chem. 1991;266(32):21718–21722. Adly MA, Assaf HA, Nada EA, Soliman M, Hussein M. Expression of nerve growth factor and its high-affinity receptor, tyrosine kinase A proteins, in the human scalp skin. J Cutan Pathol. 2006;33 (8):559–568. Pincelli C, Sevignani C, Manfredini R, Grande A, Fantini F, BracciLaudiero L, Aloe L, Ferrari S, Cossarizza A, Giannetti A. Expression and function of nerve growth factor and nerve growth factor receptor on cultured keratinocytes. J Invest Dermatol. 1994;103(1):13–18. Stefanato CM, Yaar M, Bhawan J, Phillips TJ, Kosmadaki MG, Botchkarev V, Gilchrest BA. Modulations of nerve growth factor and Bcl-2 in ultraviolet-irradiated human epidermis. J Cutan Pathol. 2003;30(6):351–357. Paus R, Luftl M, Czarnetzki BM. Nerve growth factor modulates keratinocyte proliferation in murine skin organ culture. Br J Dermatol. 1994;130(2):174–180. Marconi A, Vaschieri C, Zanoli S, Giannetti A, Pincelli C. Nerve growth factor protects human keratinocytes from ultraviolet-Binduced apoptosis. J Invest Dermatol. 1999;113(6):920–927. Marconi A, Terracina M, Fila C, Franchi J, Bonte F, Romagnoli G, Maurelli R, Failla CM, Dumas M, Pincelli C. Expression and function of neurotrophins and their receptors in cultured human keratinocytes. J Invest Dermatol. 2003;121(6):1515–1521. Pincelli C. Nerve growth factor and keratinocytes: a role in psoriasis. Eur J Dermatol. 2000;10(2):85–90. Pincelli C, Haake AR, Benassi L, Grassilli E, Magnoni C, Ottani D, Polakowska R, Franceschi C, Giannetti A. Autocrine nerve growth factor protects human keratinocytes from apoptosis through its high affinity receptor (TRK): a role for BCL-2. J Invest Dermatol. 1997;109(6):757–764. Pincelli C, Yaar M. Nerve growth factor: its significance in cutaneous biology. J Invest Dermatol Symp Proc. 1997;2(1):31–36. Wehrli P, Viard I, Bullani R, Tschopp J, French LE. Death receptors in cutaneous biology and disease. J Invest Dermatol. 2000;115 (2):141–148. Botchkarev VA, Botchkareva NV, Albers KM, Chen LH, Welker P, Paus R. A role for p75 neurotrophin receptor in the control of apoptosis-driven hair follicle regression. FASEB J. 2000;14(13): 1931–1942. Adly MA, Assaf HA, Hussein MR. Expression pattern of p75 neurotrophin receptor protein in the human scalp skin and hair follice: Hair cycle-dependent expression. J Am Acad Dermatol. 2009;60 (1):99–109. Krygier S, Djakiew D. The neurotrophin receptor p75NTR is a tumor suppressor in human prostate cancer. Anticancer Res. 2001;21(6A):3749–3755. Nakagawara A. Trk receptor tyrosine kinases: a bridge between cancer and neural development. Cancer Lett. 2001;169 (2):107–114.
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35. Peacocke M, Yaar M, Mansur CP, Chao MV, Gilchrest BA. Induction of nerve growth factor receptors on cultured human melanocytes. Proc Natl Acad Sci USA. 1988;85(14):5282–5286. 36. Di Marco E, Mathor M, Bondanza S, Cutuli N, Marchisio PC, Cancedda R, De Luca M. Nerve growth factor binds to normal human keratinocytes through high and low affinity receptors and stimulates their growth by a novel autocrine loop. J Biol Chem. 1993;268(30):22838–22846. 37. Zhai S, Yaar M, Doyle SM, Gilchrest BA. Nerve growth factor rescues pigment cells from ultraviolet-induced apoptosis by upregulating BCL-2 levels. Exp Cell Res. 1996;224(2):335–343. 38. Alleva E, Petruzzi S, Cirulli F, Aloe L. NGF regulatory role in stress and coping of rodents and humans. Pharmacol Biochem Behav. 1996;54(1):65–72. 39. Alberch J, Perez-Navarro E, Arenas E, Marsal J. Involvement of nerve growth factor and its receptor in the regulation of the cholinergic function in aged rats. J Neurochem. 1991;57(5):1483–1487.
40. Garcia-Suarez O, Germana A, Hannestad J, Perez-Perez M, Esteban I, Naves FJ, Vega JA. Changes in the expression of the nerve growth factor receptors TrkA and p75LNGR in the rat thymus with ageing and increased nerve growth factor plasma levels. Cell Tissue Res. 2000;301(2):225–234. 41. Backman C, Rose GM, Hoffer BJ, Henry MA, Bartus RT, Friden P, Granholm AC. Systemic administration of a nerve growth factor conjugate reverses age-related cognitive dysfunction and prevents cholinergic neuron atrophy. J Neurosci. 1996;16(17):5437–5442. 42. Amendola T, Aloe L. Developmental expression of nerve growth factor in the eye of rats affected by inherited retinopathy: correlative aspects with retinal structural degeneration. Arch Ital Biol. 2002; 140(2):81–90. 43. Aloe L. Rita Levi-Montalcini: the discovery of nerve growth factor and modern neurobiology. Trends Cell Biol. 2004;14(7):395–399.
Pathology
28 Pathology of Aging Skin Qunshan Jia . J. Frank Nash
Introduction Human skin is the largest and the most complex organ functioning as a physical and biochemical barrier to protect the human body from water loss as well as environmental insults including pathogens, chemicals, physical agents and solar ultraviolet radiation (UVR) throughout life. More than that, the skin provides crucial physiological functions including immune defense, thermoregulation, sensoring, endocrine as well as metabolism. Aging is a chronological process accompanied by a progressive loss of physiological function in multiple organs. Skin undergoes an aging process accompanied by physical changes, clinical manifestations and significant psychological consequences. According to recent statistics, around 25% of Americans are expected to be 65 years or older by the year 2030 [1]. Therefore, it is important to understand the chronological skin aging process and its accompanying physiological consequences for medical reasons and for the personal care industries.
Skin Development and Anatomic Structure To better understand the pathology and physiology of the aging skin, it is crucial to understand the development, anatomical structure and physiological function of normal skin.
The Structure and Function of Skin Barrier The skin is structurally divided into epidermis and dermis separated by the basal lamina. Epidermis is the major protective outer layer, with keratinocytes as the major cell population. The epidermis is a stratified epithelium derived from a single layer of ectoderm after gastrulation. Wnt/BMP signaling enables the ectoderm cells to adopt an epidermal fate instead of neurogensis by inhibiting fibroblast growth factor (FGF) signaling pathways. Over time, the embryonic epidermis will differentiate
into epidermal cells under the influences of BMP, Notch signaling, the hair placode and eventually the hair follicle induced by Wnt signaling and its downstream signaling including SHH, GLI1, PTC. The sebaceous glands are appendages of hair follicles, the origin of which remains unknown [2]. The stratified epidermis is around 100–150 m thick, which can be further divided into four distinct layers: stratum basale, stratum spinosum, stratum granulosum and stratum corneum, as illustrated in > Fig. 28.1. The stratum basale consists of epidermal stem cells, a single layer of columnar cells attached to the basal lamina via hemidesmosomes and expressing keratin(K)14 and K5. Once the cell leaves the basal layer toward the skin surface, it starts to express K1 and K10 in the stratum spinosum. The stratum spinosum, which contains lipid-enriched lamellar bodies, becomes progressively larger due to keratin synthesis and lipogenesis and finally reaches a stage called stratum granulosum with unique lamellar bodies and is ready to differentiate into corneocytes. Finally, intracellular organelles in the corneocyte undergo selfdestruction and the lipid packaged in lamellar granules (LG) is released into the intercellular space. The LGs are small organelles full of stacks of lipid lamelle consisting of phospholipids, cholesterol, glucosylceramides and several enzymes important for the lipid processing including acid hydrolases, ß-glucocerebrosidase, phospholipid A2 and lysosomal acid lipase [3]. Once released from the LGs, the short stacks of lipid membranes will reorganize and transform into an edge-to-edge fusion catalyzed by enzymes released at the same time. In the end, the stratum corneum will form the outermost seal with 18–21 cell layers 20–40 micrometer thick in human skin mainly consisting of dead corneocytes and the secreted lipids [3]. The brick and mortar structure is a classic model for the organization of the stratum corneum (> Fig. 28.2). The most important function of the stratum corneum includes prevention of water loss and percutaneous absorption of xenobiotics (> Table 28.1) [4]. During the final stages of keratinocyte differentiation, the intracellular keratins and filaggrin will interact with each other to form a condensed protein complex, transforming the epidermal
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_28, # Springer-Verlag Berlin Heidelberg 2010
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Pathology of Aging Skin
. Figure 28.1 The structure of the epidermis. The epidermis contains stratum corneum, stratum granulosum, stratum spinosum and stratum basale form outside to inside. The stratum basale consists of epidermal stem cells, a single layer of columnar cells attached to the basal lamina via hemidesmosomes. The stratum spinosum is rich with lipid-enriched lamellar bodies, become progressively larger due to the keratin synthesis and lipogenesis and finally reach a stage called stratum granulosum which has unique lamellar bodies and is ready to differentiate into corneocyte. Finally, corneocyte intracellular organelles undergo self destruction and the lipid packaged in lamellar granules (LG) is released to the intercellular space. The stratum corneum forms the outermost seal with 18–21 cell layers 20–40 micrometer thick in human skin mainly consisting of dead corneocytes and the secreted lipids. Filaggrin is a key protein required for the formation of the stratum corneum (SC) barrier which is also essential for SC hydration, as it acts as a source of hygroscopic amino acids and their derivatives, known as natural moisturizing factor (NMF)
. Figure 28.2 The simple ‘‘brick’’ and ‘‘mortar’’ model for stratum corneum. The dead corneocytes were embedded in the intercellular lipid matrix, which forms the primary barrier for the skin
Pathology of Aging Skin
28
. Table 28.1 Multiple protective functions of mammalian stratum corneum (Adapted from Elias PM. [4]) Function
Principal compartment
Structure basis
Chemical basis
Permeability
Extracellular matrix
Lamellar bilayers
Ceramides, cholesterol, nonessential fatty acids in proper ratio
Antimicrobial
Extracellular matrix
Lamellar bilayers
Antimicrobial peptides, FFA, sph
Antioxidant
Extracellular matrix
Lamellar bilayers
Chol, FFA; secreted vit. E, redox gradient
Cohesion (integrity)
Extracellular matrix
Comeodesmosomes(CD)
Intercellular DSG1/DSC1 homodimers
Mechanical/ rheological
Corneocyte
Cornified envelope; keratin filaments
g-Glutamyl isopeptide bonds
Chemical (antigen exclusion)
Extracellular matrix
Extracellular lacunae
Hydrophilic products of CD
Psychosensory interface
Extracellar matrix
Lamellar bilayers
Barrier lipids
Hydration
Corneocyte
Cytosol
Filaggrin proteolytic product; glycerol
Electromagnetic radiation
Corneocyte
Cytosol
Cis-urocanic acid (histidase activity)
Initiation of inflammation
Cornocyte
Cytosol
Proteolytic activation of pro IL-1a/b
cell into a flatten corneocyte. The ‘‘bricks’’ are the multiple layers of protein-enriched dead corneocytes tightly packed and surrounded by a very dense cross-linked protein structure called the crucified envelope. This protein envelope is cross-linked with the surrounding lipid envelope, the ‘‘mortar’’, which is made up of hydrophobic lipid lamellae in the intercellular regions and gives the skin physical protection against water and other molecules [5]. Multiple genes have been identified which play important roles in skin barrier function. For example, aberrant expression of filaggrin will result in multiple barrier defects from atopic dermatitis to flaky skin [6]. Involucrin, loricrin and trihohyalin are major protein components on the surface of the corneocyte forming a protein envelope cross-linked by transglutaminases. Excessive loss of water and neonatal death has been observed in transglutaminase-deficient mice, illustrating the importance of the stratum corneum’s barrier function during development [7]. The specific cell–cell junction between stratum corneum and stratum granulosum, named desmosomes, is also critical, the loss of which disrupts the stratum cell adhesion resulting in barrier function defects [8]. The lipid composition of the stratum corneum is critical for barrier function. Studies have shown that the
stratum corneum mainly consists of equimolar ratios of ceramides, cholesterol and free fatty acids. Ceramides are crucial in the formation of the lipid envelope and a deficiency is associated with atopic dermatitis [9]. Cholesterol is synthesized by the epidermis and is important for intermixing different lipids. The cholesterol efflux is regulated by a membrane transporter named ATP binding cassette subgroup 1 member transporter or ABCA12. Failure of different enzymes involved in cholesterol synthesis results in significant epidermal barrier defects [6]. Finally, essential fatty acids deficiency will also result in a red, rough skin with significant transepidermal water loss [10, 11] and disruption of gene coding for fatty acid transport protein 4 (FatP4) has been found to result in neonatal death due to disturbed skin permeability [12]. The nucleated lower layers of the epidermis are important for mediating barrier function. The entire loss of the epidermis will lead to life-threatening water loss and rampant microbiological infection, compared to the relatively mild consequences associated with stratum corneum damage [6]. Multiple cell–cell tight junction proteins such as claudins, zonula occludens protein-1, and multi-PDZ protein-1 have been found in the stratum granulosum and the upper stratum spinosum. The
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Pathology of Aging Skin
importance of these tight junctions is illustrated by extreme water loss in mice with claudin-1 and E-cadherin deficiency. Adherens and desmosomes are a group of junction proteins essential for stabilizing the cell–cell adhesion in the living layer of the epidermis. Reducing desmoglein-3 and E-caldherin will result in a barrier defect leading to either leaky tight junctions or a lethal dehydration [6]. The intercellular gap junctions are important channels for cell–cell communication. Disturbing such gap junctions, found in connexin43 loss of function mouse studies, results in barrier defects [6]. Keratins are the major component of intra-keratinocyte filament web which interacts with the junction proteins, desmosomes and hemidesmosomes. Homozygous K10 knock-out mice will develop an extremely delicate epidermis while heterozygous K10 mice will show impaired barrier function repair and skin hydration defects, demonstrating the importance of this differentiation-related protein’s effect on epidermal barrier function [6].
Epidermis Homeostasis and Turnover In healthy skin, the epidermis replenishes itself and maintains a status of homeostasis in response to physical and chemical challenges. In humans, the turnover rate for the epidermis is approximately 4 weeks. During this process, cells in the stratum basale divide and move upward while undergoing differentiation during this progression. Both symmetric and asymmetric models have been used to describe this process. In the symmetric model, the stratum basale daughter cells with lower levels of integrin after mitosis will detach and begin the journey to becoming part of the stratum corneum. Daughter cells with higher levels of integrin will keep their proliferating potential and stay attached to the basement membrane. In the asymmetric model, the mitotic epidermal cells divide disproportionately so that the attached daughter cells receive more integrin and growth factors to keep them attached to the basement membrane and proliferating while the detached suprabasal daughter cells receive less or dissimilar signals such as Notch, leading to differentiation and commitment to stratum corneum replacement [2]. Proliferation and growth of cells in the stratum basale must be tightly controlled to ensure the normal function of the stratum corneum. Recent studies have shown that the extracellular matrix (ECM) and growth factors residing in the basement membrane play a crucial role in controlling cell proliferation in the stratum basale. Two
types of junctions are responsible for the cell–ECM connection. The hemidesmosomes containing a6b4 integrin and the focal adhesions containing a3b1 integrin control growth and migration through a physical interaction with kinases such as Ras/MAPK signaling. The E-caldherin in cell–cell junctions also contributes to basal cell proliferation and migration through its association with regulatory proteins such as Rho-GTPase. Although the exact mechanisms are unclear, it is accepted that the adherens junction can serve as a signaling center and sense the epidermal cell density and thus provide a feedback loop through specific signaling kinase pathways to control cell activity and proliferation [2]. The epidermal barrier function is also maintained by multiple signaling pathways as indicated by studies of acute skin disruption. For example, proinflammatory cytokines including interleukin-1, interleukin-6 as well as tumor necrosis factors have been observed following acute barrier damage. As well, knocking down these genes will delay barrier formation after acute disruption [6]. Recently, a calcium gradient was identified in the epidermis with the highest calcium concentration in stratum granulosum and the lowest concentration in stratum corneum. Calcium is crucial for lamellar body exocytosis and epidermal protein synthesis at the later stage of keratinocyte differentiation and migration in response to barrier permeability damage. In addition, calcium plays important roles for transglutaminase I activity and cell–cell adhesion during epidermal differentiation. The relationship between the calcium level and the transmembrane water loss has been observed in Darier’s and Hailey-Hailey diseases caused by the defects of a gene encoding a calcium transporter [6]. Another signaling pathway important in maintaining the barrier is 3’5’-cyclic adenosine monophosphate (cAMP), a secondary messenger in multiple systems. Biochemistry studies have revealed a reverse relationship between the cAMP level and the barrier recovery after acute epidermal damage. Vascular endothelial growth factor (VEGF) generated by the epidermis is also required for the integrity of the barrier function with homozygous VEGF knock-out mice showing impaired permeability barrier homeostasis after acute repeated tape striping. Finally, a new mechanism has been suggested for maintaining epidermal homeostasis in which both the lipid matrix and corneocytes are involved. In general, the disruption of stratum corneum will inevitably increase cytokines and calcium concentrations within the epidermis, which in turn will promote lipid synthesis and secretion into the intercellular matrix to facilitate barrier recovery. At the same time, epidermal cornification will be enhanced by serine protease and caspase-14
Pathology of Aging Skin
signaling turned on by protease activated receptor type 2 (PAR2) in response to acute barrier defects [13]. In addition to the keratinocytes, several other cellular populations have also been found in the epidermis including Langerhans cells, Merkel cells, and melanocytes. Langerhans cells are dendritic cells residing in the epidermis involved in antigen presentation and immunoserveillance [14]. Melanocytes are cells derived from neural crest responsible for skin pigment and hair color through melanin production. They are usually found in the basal layer of epidermis and in hair follicle. The Merkel cells are also derived from the neural crest cells and are associated with the sensory nerve endings. The dermis, located below the epidermis basement membrane, is rich with blood supply providing nutrients and circulatory support to the epidermis. In humans, the boundary between the epidermis and dermis undulates due to epidermal protrusions into the dermis resulting in the ‘‘rete pegs’’. The cellular populations account for 10% of the dermal content including the fibrocyte, monocyte, histiocyte, Langerhans cells, lymphocytes, and eosinophils, along with the vascular- and lymphatic-associated cells while the remaining 90% is mainly connective tissue matrix made up of Type I collagen, elastic fibers, and blood vessels. The collagen in the dermis provides mechanical protection to the body as well as the shape and form by holding all structures together. The blood plexus provides oxygen and nutrients to the living part of the epidermis and removes waste products of metabolism from the epidermis. Body temperature regulation through control of blood flow and sweating is also achieved by dermis as well as the sensations of touch, pain, heat and cold through the neural fiber embedded in the dermis [15].
Skin Physiology Immune Function While the skin acts as the principle barrier to protect the body from the water loss, it also serves as a non-specific defense against infections. Phagocytic cells such as neutrophils, macrophages and natural killer cells (NK) present in the skin play a significant role in defense against pathogens. Meanwhile the complement system and cytokines turned on by components of pathogens, e.g., LPS, also contribute to the innate immune response of the skin. Both the innate and adaptive immune responses are equally critical to the host defense against foreign invaders. The skin possesses important peripheral components such as Langerhans cells (LC) and extravasated lymphocytes from
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circulation for adaptive immune responses. The dendritic LC will process and present an antigen to the resident T lymphocytes which will initiate a cell-mediated acquired immune response [16–19]. Generally, the skin will initiate a rapid cytokine-driven cutaneous inflammation in response to keratinocyte injury followed by a specific adapted immune response to foreign antigens with high specification and immune memory. Recent studies have shown the epidermal keratinocyte cell–cell junctions will promote the immune response after the skin is injured. It has been found that b-catenins in cell–cell junctions function not only as adhesion molecules but also as transcription regulators. Through unknown mechanisms, p120 catenin and a-catenin can affect the transcriptional activity of NF-kB to induce cytokines. It is hypothesized that keratinocyte injury will trigger the catenin/NF-kB signaling cascade, inducing cytokines, chemokines which will recruit immune cells to initiate the innate immune response first, and adapted immune response later. Once the epidermis is repaired and the cell–cell junction returns to normal, the inflammation will diminish. This is supported by the findings that chronic inflammation will develop after a-catenin and p120 are mutated [2, 18]. It has also been found that acute barrier disruption not only increased hapten infiltration but also promoted skin immune function by promoting the production of cytokines on epidermal cells and upregulation of co-stimulatory molecules on Langerhans cells. Thus, the intact skin barrier function is of importance for skin immune function.
Skin Metabolism In addition to its physical barrier, the skin also provides a biochemical barrier by virtue of enzymatic activities in epidermis and dermis. This enzymatic and biochemical barrier enable the skin to biotransform topically applied xenobiotics and to function as an important extrahepatic organ. Most of the metabolizing enzymes are located in the epidermis [20]. For example, cytochrome P-450 is present in the skin and responsible for the oxidative metabolism of steroid hormones including androgens, estrogens, progesterone and glucocorticoids, as well as fatty acids. As the major Phase-I enzyme, P-450 plays a critical role in metabolic detoxification or, in some cases, activation. The presence of Phase-II conjugating enzymes such as glucoronyltransferase, sulfotransferase and glutathioneS- transferase enable the skin to further detoxify and eliminate the metabolites generated from Phase-1 reactions. The major enzymes found in the skin and involved in metabolism are presented in > Table 28.2 [21]. An
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. Table 28.2 Classes of enzymes detected in skin (Adapted from Smith et al. [21]) Phase I enzymes activaton/ functionalization mechanisms
Function
Microsomal mixed function oxidase
Hydroxylation
Cytochrome P450
Alcohol oxidation
NADPH dehydrogenase
Epoxidation N-, O- and S-dealkylation oxidative deamination N- and S-oxidation Dehalogenation Reduction of azo and nitro compounds Ring cleavage of heterocyclic ring systems
Alcohol dehydrogenases
Interconversion of alcohols and aldehydes
Aldeyde dehydrogenases
Conversion of aldehydes to acids
Flavin-containing monooxygenases
Oxidation of secondary and tertiary amines Oxidation of imines and arylamines
Esterases
Ester hydrolysis to yield acid and alcohol
Amidases
Amide hydrolysis
Phase II enzymes and conjugation reactions UDP-glucuronosyltransferase
Glucuronidation
Sulphotransferase
Sulphation
Methyltransferase
Methylation
Acetyltransferase
Acetylation
Glutathione S-transferase
Glutathione conjugation at epoxides and halides
Miscellaneous reactions condensation
Amino acid conjugation
example of metabolic activation is the enzyme alcohol dehydrogenase which is present in keratinocytes and involved in the transformation of the innocuous chemical cinnamic alcohol into cinnamic aldehyde, a potent skin sensitizer. Finally, a recent study reported that the metabolic rate in the skin affects dermal penetration of lipophilic compounds with a higher metabolic rate generally correlated with greater penetration [22].
Skin Sensory and Thermoregulation Advances in immunochemical staining have revealed the presence of neuronal fibers in the epidermis. More than 90% of these are small-diameter, unmyelinated C-fiber and/or thinly myelinated A-d fibers located in the border between of the basement membrane and epidermis. The major functions of cutaneous neuronal fibers are sensory and integration of incoming signals for pain, itch and other stimuli. The epidermal nervous system possesses important efferent paracrine and trophic functions that affect cutaneous cells, immune cells, and other axons. This is supported by findings that nociceptive nerve endings have close contacts with LC, mast cells and even keratinocytes. The trophic effects of this innervation were identified in studies where nerve endings were cut resulting in a reduction of the keratinocyte mitotic rate and a ‘‘shiny atrophic’’ skin. In addition, most dermal vessels, eccrine sweat as well as arrector pili muscles are densely innervated by the neuron fibers [23]. Thermal regulation of the human body is controlled by receptors located in the skin called transient receptor potential (TRP) superfamily which are cation-selective ion channels consisting of six transmembrane subunits. TRPs are activated within a specific temperature range in response to the environmental temperature changes. With the cooperation of central nerve system, healthy individuals will get rid of excess heat by sweating and vasodilation when it is too hot and minimize heat loss through vasoconstriction, and increase heat generation by shivering when it is too cold. The sweat glands dispersed within the skin control body temperature by energy demanding sweat evaporation. Up to 2 L of sweat can be evaporated in an hour mainly by the eccrine glands. This control for body temperature through sweat production is important for thermal maintenance [24].
The Structural and Physiological Changes in Aged Skin Chronological aging is an inevitable biological process leading to structural and functional changes during the lifespan of all organisms. This programmed route is inherently determined by genetics and is significantly affected by multiple environmental factors. Dryness, wrinkles, irregular pigmentation are the primary visual findings of aging skin in human beings accompanied by histological changes ranging form impaired stratum corneum replenishment to a flattening of the dermal–epidermal junction, a marked elasticity loss and atrophy of the dermal
Pathology of Aging Skin
. Table 28.3 Characteristic of intrinsic aging (Adapted from Farage et al. [88]) Characteristic
Intrinsic aging
Overall Metabolic processes
Slow down
Clinical appearance
Smooth unblemished, loss of elasticity, fine wrinkles
Skin color
Pigment diminishes to pallor
Skin surface marking
Maintains youthful geometric patterns
Onset
Typically 50s–60s (woman earlier than men)
Severity
Only slightly associated to degree of pigmentation
Epidermis Thickness
Thins with aging (not consistent)
Proliferative rate
Lower than normal
Keratinocytes
Modest cellular irregularity
Dermoepidermal junction
Modest reduplication of lamina dense
Dermis Elastin
Elastogenesis followed by elastolysis
Elastin matrix
Gradual decline in production of dermal matrix, only modest increase in the number and thickness of elastic fibres in the reticular dermis
connective tissue due to a reduction and disorganization of its major extracellular matrix components [25–27]. The characteristic histological changes and its impacts on barrier function during aging will be reviewed (> Table 28.3).
Anatomic Changes in Aging Skin Epidermis The overall histology change in epidermal thickness is neither obvious nor consistent with the conclusion that this structure is ‘‘thinner’’ in aged skin [28]. There is general agreement, however, that the intersection of the epidermis and dermis is flattened in aged skin with a correspondingly diminished connecting surface area leading to increased fragility and reduced nutrient transfer between the dermal and epidermal layers. A significant, 30–50%, decrease in
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epidermal turnover rate has also been observed in the third to eighth decades of life. Such an impaired epidermal turnover rate might account for some of the pathological findings in aged people [15, 25, 26]. Another significant change in the epidermis of aging skin is in the amount of glycosaminoglycans (GAGs), the primary constituents of skin which hold water up to 1,000 times their weight. Hyaluronic acids (HAs) are a major type of GAGs and are produced by fibroblasts in the dermis and keratinocytes in the epidermis. In the epidermis, HAs are localized in the epidermal intercellular spaces at the middle spinous layer. The total amount of HAs is reduced significantly in the epidermis which may contribute to the reduced water binding and the visible changes of the aged skin, including wrinkling, altered elasticity, reduced turgidity and diminished capacity to support the microvasculature of the skin. However, the skin of older subjects has a comparable level of HAs in dermis compared to younger ones [15, 26].
Dermis In contrast to the epidermis, a consistent lost of 20% dermal thickness has been observed in aged skin, characterized by reduced cellular components and vascular networks. For example, in aging skin a reduction of collagen due to decreased synthesis by fibroblasts has been observed. As the primary structural component and the most abundant protein found in dermis, collagen is responsible for conferring strength and support to the structure of human skin. In aged skin, the collagen organization is characterized by disarrayed thickened fibrils in rope-like bundles in comparison to the more structured pattern observed in younger skin. The ratio of collagen types also changes with age due to the loss of collagen I in aged skin. Overall, the collagen content per unit area of skin surface is known to decline approximately 1% per year. The loss of collagen in intrinsically aged skin will lead to an epidermal and dermal atrophy characterized by flattening of the rete ridges and subsequent wrinkle formation (> Table 28.3). The accumulation of broken elastic fibers has been observed as well. Elastin is a connective tissue protein that allows many tissues in the body to resume their shape after stretching or contracting. An accumulation of amorphous elastin material has been associated with aging and attributed to the increased level of matrix metalloproteinases which are thought to play a role in elastin degradation. The aging dermis has a reduced vascular network in comparison to young skin which will result in reduced blood flow, impaired nutrient exchange,
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inhibited thermoregulation, and decreased skin surface temperature. Melanocytes which can synthesize melanin to protect the skin from UV light are also significantly reduced with age by a rate about 8–20% per year. For this reason, sun protection remains critically important for elderly patients [15, 25–27].
The Barrier Function Change of the Aged Skin Basal trans-epidermal water loss (TEWL) has been measured in human and mouse to assess the impact of aging on skin barrier function. The results have revealed no significant change in TEWL in aged subjects (>80 years) compared to the young adult (<30 years) with similar results in mice [29]. Further analysis found no difference in the quantity of laminar bundles in granular cells or the composition of fatty acids in stratum corneum. There was, however, a reduction in total lipid level by 30% [29, 30]. In spite of the seemingly normal TEWL in older subjects under basal conditions, both barrier function and stratum corneum integrity defects are observed in aged epidermis when damaged. For example, the number of tape strips required to perturb the barrier function is about 18 for aged human subjects compared to a 30 for younger subjects [29]. The time for acetone-induced barrier perturbation is less than 10 min in age subjects compared to 30–60 min in young human subjects. In addition, the barrier function recovery rate is also significantly delayed in older human subjects and hairless mice models compared to their younger cohorts after acute tape stripping. These studies indicate that differences in barrier function in the epidermis of older people may only emerge following barrier disruption.
pH and Barrier Function in the Aged Skin Recent studies have found that stratum corneum pH is essential for epidermal barrier homeostasis. In this regard, a significantly reduced level of stratum corneum lipids might be the biochemical basis for the defects of aged epidermis [29–31]. Optimally, an acidic environment is needed for lipid processing enzymes such as ß-glucocerebrosidase and spingomyelinase to function after LB secretion following acute epidermal disruption. This association has been confirmed in studies showing delayed barrier recovery at neutral pH and a defective dynamic barrier in neonate
stratum corneum with a surface pH around 7.0 [32–34]. Inhibition of the Na+/K+ antiporter, NHE1, and sPLA2 will significantly delay postnatal acidification of the skin. These studies suggest that acidic pH is mainly established by endogenous phospholipase and NHE1 through hydrolyzing membrane phospholipids and generating acidifying free fatty acids [34, 35]. Stratum corneum acidification has been reported to be impaired in moderately aged human subjects with surface pH in unperturbed skin increasing from 5 to around 5.5. This phenomenon has been observed in the corresponding aged mice where surface pH increased from around 5.5 to 6. With the help of fluorescence imaging, pH in aged mice was found increased throughout the whole stratum corneum compared to their young control groups. To assess the impact of pH on barrier recovery, TEWL was measured after acute acetoneinduced barrier disruption. The results showed a significant 6-h delay in the moderately aged mice after barrier disruption compared to their young counterparts. Interestingly, this difference in barrier recovery was reversed to the level observed in a younger group by a single application of lactobionic acid. Collectively, these data suggest that the impaired stratum corneum acidification may account for epidermal barrier defects observed in aged subjects. Both morphologic and biochemical studies have shown that epidermal lipid synthesis was similar in moderately-aged and young individuals based on comparable levels of LB in epidermis and mRNA for key enzymes responsible for lipid synthesis. However, a defective lipid processing was observed in the analysis of moderate-aged subjects’ epidermis in contrast to the total lipid synthesis and secretion defects observed within the epidermis in advanced age, i.e., >70 years old [36]. A delayed formation of mature lamellar membrane after 6 hours of disruption was found at the SG-SC boundary in the epidermis of moderately aged subjects accompanied by diminished BGC activity revealed by in situ zymography assay. In addition to the lipid processing defects in the epidermis, an increased pH has been proposed to activate a different family of enzymes, namely the serine proteases, which will degrade the cell–cell junction protein corneodemosomes (CD) disrupting the epidermal integrity in moderately-aged subjects. Histological studies in the epidermis of moderately-aged subjects revealed a significant correlation between a decrease of CD and the impaired SC integrity. The co-location of NHE1 with the BGC and serine proteases at the SC–GC junction and the decrease of NHE1 activity within the moderately aged epidermis
Pathology of Aging Skin
suggested that NHE1 might be responsible for the agingrelated pH increase and barrier defects in moderately-aged epidermis. NHE1 is the only sodium-proton exchanger class of non-energy-dependent transporters expressed in keratinocytes and in epidermis, which has been shown to affect the intracellular pH. Recent studies indicated that NHE1 is located at the SG–SC boundary and deletion of which will reduce SG–SC acidification and impair SC barrier function recovery as well after acute tape stripping. In addition, an altered SC lipid processing and defects of lamellar membrane maturation has been observed in NHE / epidermis [35, 37]. Taken together, these data suggest that the age-related NHE1 down-regulation may, in part, account for the pH abnormality found in aged epidermis.
Peroxisome Proliferator Activated Receptors (PPARs) The down regulation of PPARs with age might also contribute to a defective barrier in aging skin. PPARs are a group of nuclear receptors that heterodimerize with RXR and are activated by fatty acids, prostaglandins, eicosanoids as well as other lipid metabolites. Based on in situ hybridization studies, PPARa and g are expressed in epidermis. Topical application of PPARa activators accelerates SC acidification, which, in turn, significantly improves SC barrier function and epidermis integrity by enhancing LB secretion and lipid processing [38]. It has been reported that serine phospholipase A2 (sPLA2) was activated by PPARa activator, and simultaneous treatment of with PPARa and sPLA2 inhibitors will reverse PPARa induced SC acidification suggesting PPAR/sPLA2 might be important signaling pathways for SC pH regulation [38]. Very interestingly, PPARa mRNA level decreased about 30% in rat kidney (25 months) compared to their young controls (13 months). Similarly, significantly, 53% and 64% decreases of PPARa nuclear protein level and DNA binding activity in kidney extract, respectively, was observed in older rats. Although the epidermal expression of PPARa in older rats was not tested, a similar trend would be expected. If this is true, it might help to explain the increased pH level in the epidermis of aging skin. However, in one study, it was fount that PPARa knock-out did not affect the SC’s pH and its barrier function in mice. While knocking out PPARg would significantly impair the SC barrier function, no age-related decline of PPARg level was noticed in aged epidermis [38–40]. So it would be
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very interesting to clarify the relationship between the PPARs, SC pH and barrier function in future studies.
The Psychological Stress, Estrogens and Barrier Function in Aged Skin Epidermal barrier function can be affected by psychological stress-induced glucocorticoid (GC) secretion. Previous studies have shown that long-term treatment with GC will reduce epidermal proliferation resulting in an atrophy of the skin. Shorter duration of treatment with GCs will block epidermal lipid production which delays lipid secretion and lamellar body formation resulting in impaired epidermal barrier function. This effect was reversed by topical application of lipids [41, 42]. Indeed, when compared with healthy young subjects, it is conceivable that the elderly are more ‘‘stressed’’, accompanied by activation of the hypothalamus–pituitary–adrenal (HPA) axis and an increased GCs which might contribute to abnormalities of epidermal barrier function in older people [41]. It has been recognized that the serum estrogen level is also important for the maintenance of skin function in females. Age-related loss of estrogen will affect skin collagen content, dermal thickness and elasticity as well as water content and might result in an impaired skin barrier. Although a recent study showed estrogen will promote epidermal mitotic activities, the exact mechanism(s) by which estrogen influences skin barrier function is unclear [43–46].
Immune Function Change in Aged Skin The increased prevalence of skin infections in older subjects is suggestive that aging is associated with reduced peripheral immunity [19]. Physical barrier function defect, increased pH, skin dehydration, elevated GC level and accumulated oxidative stress products all contribute to the suboptimal function of the skin immune system in aged human beings. At a cellular level, no significant differences in Langerhans Cell abundance or localization between young and aged skin were noticed. However, the pan-haematopoietic cell antigen CD45 positive cells were found to be reduced and there was a significant loss of T cells and atypical dendritic epidermal T cells (DETCs) in murine aging skin suggesting an impaired immune response [47].
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Metabolism There have been few studies examining aging skin metabolism. In general, hepatic-mediated enzymatic activities decline during chronological aging. A similar trend might be expected in the skin. The effects of aging on skin metabolism are mainly affected by the physiological and pathological factors during the aging process. Chief among these is oxidative stress. Due to the attenuated ability to degrade oxidized proteins during aging, the accumulation of such products might further disturb cellular metabolism. At the same time, the functioning metabolic enzymes might also be attacked by free radicals [48]. Oxidative stress will also damage keratinocyte mitochondrial function, which forces the cell to switch the balance to anaerobic glycolysis for compensation [49]. In addition, cutaneous blood vessel networks have been found to be decreased due to the angiogenesis defects in the aging skin. Therefore a decrease of metabolic rate will be expected because of the suboptimal level of oxygen [50].
Sensory Function and Thermal Regulation in Aged Skin As mentioned, skin sensory innervation systems consist of free nerve endings located in the dermis and epidermis which play an important role in skin homeostasis. The function of these sensory nerves is associated with immune function, inflammation, wound healing, thermoregulation, and hair growth. Protein gene product 9.5 (PGP 9.5) immunostaining studies have shown an ageassociated decrease in skin sensory innervation networks. In chronological aging, a decrease in skin innervation is associated with the loss of neuronal networks around the sweat glands, and a decrease in perception of thermal stimuli and tactile sensation. However, painful sensation is relatively conserved [51]. Both the adrenergic vasoconstrictor system and an active vasodilator system work in concert to achieve the skin thermoregulatory by adjusting skin blood flow. Skin blood flow will increase as the core body temperature increases and, sweating and cutaneous active vasodilation (AVD) will occur to increase the evaporation once a threshold is reached. Reflex vasoconstriction (VC) of cutaneous blood vessels in response to cooling will effectively minimize heat loss. It has been found that there is a reduction in the ability to raise skin blood flow during heat stress in older subjects. Evidence from heat stress studies have shown that the attenuated cutaneous reflex vasodilation is mainly due to the attenuated NO-mediated pathways in older (71 6 year)
compared to the young (23 2 year) subjects [52, 53]. On the other side, the ability to reduce skin blood flow in response to cooling is also compromised in advanced age which pose a health risk for older in response to cooling situation. Recent studies have shown that the nonnoradrenergic-mediated VC mechanisms are mainly responsible for the aged related defects in reflex cutaneous VC [54, 55].
The Possible Molecular Mechanisms for Skin Aging Telomere and Skin Aging It is well recognized that cellular proliferation is accompanied by a progressive chromosome shortening of the telomere structure at the end of chromosome. Telomeres are the tandem repetitive DNA sequences at ends of mammal chromosomes which generally consist of several thousand base pairs with the 30 strand overhanging by 75–300 bases, i.e. (TTAGGG)n. The major function of the telomeric repeats is to protect the chromosome by shortening during cell proliferation. Recent studies have revealed telomere decrease with every cell division, which in all likelihood is inversely related to the individual’s physiologic age. At the same time, the single-stranded telomere overhang can also form a loop structure by telomeric repeat binding factor 2 (TRF2) to provide further protection of DNA integrity. Loop disruption will result in digestion of the overhanging sequence and various DNA damage responses including cellular apoptosis and senescence. This may occur naturally after critical telomere shortening or DNA damage. Dominant negative TRF2 transfection will disrupt the telomere loop structure and promote cell senescence. As the telomere reaches a threshold length after proliferation, the cells may become old and lose normal physiologic function. It has been reported that the telomere length plays an important role in controlling the age-associated transcript profile and cellular capacities during aging [56–59]. The length of telomeres is dynamic and maintained by ribonucleoprotein enzyme telomerase which can lengthen the terminal regions of telomeric DNA by addition of tandemly repeated telomeric sequences. The teleomeric balance between lengthening and shortening is influenced by genetic, developmental and physiological factors [60, 61]. For example, oxidative stress can accelerate shortening of the telomere in human fibroblasts probably through the accumulation of single-strand DNA breaks. An increase of the glutathione peroxidase (GPX-1) and copper–zinc
Pathology of Aging Skin
superoxide dismutase (CuZnSOD) mRNA in human fibroblasts will decrease the rate of telomere shortening. In an epidemiology study, a continuous loss of the telomere DNA during the aging process was observed in individuals ranging from 0 to 90 years old in peripheral hematopoietic cells suggesting a negative correlation between chronological aging and telomere shortening [62]. In addition, extensive studies in humans have revealed a correlation between telomere shortening and aging in multiple cell types including peripheral lymphocytes, fibroblasts, brain, esophageal mucosa, gastric mucosa, mixed large and small intestine mucosa, large bowel mucosa, kidney and liver [63, 64]. The relationship between telomere shortening and skin aging has also been observed in several studies. For example, analysis of DNA samples from sun protected epidermis obtained from 52 subjects ranging from 0 and 101 years old in Japan showed a rate of 36 bp reduction per year in epidermis with the estimated telomere lengths in the epidermis around 13.3 kb at birth. In another study, the length of the telomeric TTAGGG repeat sequences in skin sample from 21 human subjects between 0 and 92 years of age was measured. The results showed a statistical reduction of telomere length by a rate of 19.8 bp/year [65]. Telomere length was also measured in the skin of nine elderly patients (age range 73–95 years). The average length of telomere with this group is about 7–8 kb (7,792 596 bp). An inverse relationship between the age and telomere length was also detected in skin specimens with an average of reduction about 79 bp [65]. The yearly telomere reduction rate in the epidermis ranges from 19–75 bp a year. It did not match with the high turnover rate of the epidermis which is roughly every 4 weeks. So, in skin, the lengthening of teleomers may exist and possibly predominate. It has to be mentioned here that the epidermis telomere length measured in above studies never fell below the critical size as 5–6 kb identified in previous studies even in advanced age subjects. However, since the current technology can not measure the exact telomere length in single cells and for a single chromosome, it can not be excluded that the telomere either in a sub-population of the cells or a subgroup of the chromosome is shorter than the critical size. So, how the telomere shortening affects cell senescence remains to be investigated [63–65]. Other evidence supporting the telomere shortening and aging process comes from the telomerase activity studies [64]. Telomerase is a ribonucleoprotein DNA polymerase complex including the protein telomerase reverse transcriptase (TERT, or hTERT in humans) and a catalytic RNA (TERC). Generally, telomerase activity is
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very low in most normal human somatic cells because of the lack of expression of TERT. Previous studies have shown a decrease in telomerase activity in response to life stress and a lower telomerase activity in response to chronic emotional stress. A recent life style intervention study also revealed an increase in telomerase activity over a 3-month period after decrease of physiologic stress. In the same study, a relation between the diet associated LDL cholesterol and the telomerase activity has been observed. All these findings are suggesting a decrease of the telomerase activity in the epidermis as the age progresses [66]. Although the normal epidermis contained no or only slight telomerase activity determined by the TRAP assay, epidermal cells reserve the potential to increase telomerase activity in response to stressors such as UV light to maintain chromosome stability. For example, only one of seven specimens from sun-protected epidermis in adults showed detectable human telomerase RNA, whereas the epidermal basal cells in all samples obtained from sunexposed areas showed moderate human telomerase RNA signals. Thus, sun-exposed skin contains higher levels of telomerase activity than sun-protected skin which suggests telomerase is expressed by epidermal cells and can function as a protective mechanism in response to environmental stresses. Although age related decrease in telomerase activities has been observed in previous studies, it would very interesting to confirm this relationship in epidermis with more robust experimental data in the future. Generally, basal cells in normal epidermis have been reported to possess telomerase activity [63, 64]. In vitro studies have identified a telomere senescence characterized by enlarged cell morphology, activation of lysosomal b-galactosidase and increased inflammatory transcript profile. Dermal fibroblasts without telomerase will reach senescence after 90 population doubling (PD) cycles characterized by shorter telomere and reduced replicative potential. Gene array studies have shown an increase in pro-inflammatory genes such as p21, MCP1, IL-1, IL-15, ICAM-1, tPA, stromelysins, which might be responsible for the age-related cellular matrix degradation. However, dermal fibroblasts transfected with hTERT showed higher level of telomerase, normal length of telomere and a similar replicative potential as those from younger subjects at stage PD 20. At the same time the altered gene expression pattern was also rescued to the normal level. To identify the telomerase effects of senescent dermal fibroblast on the dermal integrity, a dermal reconstitution system was employed. The results showed that the senescent fibroblast will show a intermittent splitting between the dermis and epidermis junction in
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skin reconstitutions, while the transfected fibroblasts show a tight, intact junction, suggesting the function of telomerase for the integrity of the skin structure [63, 64]. In a recent study, a constitutively expressed telomerase reverse transcriptase (TERT) construct was introduced into a mice model to address telomerase activity on skin function during aging. The transgenic mice expressed a high level of TERT compared to their controls and at the same time they had a decreased incidence of degenerative inflammatory pathologies in skin, a better preservation of both the thickness of the epidermis and of the subcutaneous fat layer compared to their corresponding controls at the advanced age, suggesting telomerase’s anti-aging effects on the maintenance of the skin epithelia [67]. Recently, p53 and its down stream transcriptional target cycline-dependent kinase inhibitors p21/SD11 and p16 ink4a have been shown to be involved in the telomereinduced senescence in aged human fibroblast cells. At the same time, activated p21/SD11 and p16 ink4a will block the pRb phosphorylation which will lead to the programmed cell aging [68]. Disruption of cellular telomere structure will trigger on p53 and p21/SD11 pathways which in turn will induce a growth arrest and increase the intracellular level of reactive oxygen species as well. It has been found that the mouse embryonic fibroblasts show a higher superoxide anion and hydrogen peroxide production and lower catalase activity due to the deficiency of the telomerase activity which can be rescued by the telomerase restoration [69]. Although, more data are needed to clarify how the telomere/telomerase activity affect the skin barrier function during the progress of aging, all current data suggest a tight correlation between the normal skin function and the skin telomere integrity and telomerase activity.
DNA Repairing Potential in Aging Skin DNA repair capacity is impaired in primary dermal fibroblasts of older subjects. Cells possess DNA repair mechanisms to remove damaged segments mainly through nucleotide excision repair (NER) pathway or base excision repair (BER) pathway during the G1 and G2 phase, otherwise the cell will undergo apoptosis to protect the organism from potential cancerous transformation. In a UV-induced model, the cell repair capacity in the primary dermal fibroblast cell was compared between young and old subjects. The results showed that initially, UV exposure will induce similar levels of DNA damage in both groups and this will remain at 20% after
6 h in ‘‘young’’ group. In contrast, the residue level remains high, around 60–80%, in the aged subjects suggesting there is a significant difference in terms of the dermal DNA repair capacity between the young and old groups. At the same time, FACS assay revealed a significant decrease in S phase population in aged dermal fibroblast cells, suggesting that the cellular replicative potential is impaired in aged dermal fibroblast cells. However, it is surprising to find no changes in cellular antioxidant system in aged fibroblast compared to the young ones [70]. In a similar study, a lower DNA repair capacity for strand-breaks has also been observed in aged dermal fibroblasts in response to acute oxidative stress [70, 71]. Overall the lower repair capacity might account for accumulated DNA damage found in skin of older subjects and this might result in the chromosome unstability, cellular growth arrest, apoptosis as well as the chronic dermal inflammation induced by the oxidative stress.
Oxidative Stress and Skin Aging It has been very well accepted that the oxidative stress is one of the most important driving factors in the aging process [72]. Both endogenously and exogenously generated free radicals will produce oxidative damage to cellular components including DNA during the life time, which in turn will accumulate and disturb normal cellular function. To maintain normal cell function, cells are also equipped with antioxidant defenses including nonenzymatic antioxidants such as glutathione and several enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidases (GPx) to scavenge the ROS. Generally, reactive oxygen species (ROS), such as superoxide anion radicals can be formed during mitochondrial respiration and phagocytosis which will be transformed into H2O2 and hydroxyl radicals after a series of enzymatic reactions. Hydroxyl radical is capable of oxidizing any macromolecule such as lipid, DNA and protein resulting in oxidative damage. The hydroxyl radical is known to react with purine and pyrimidine bases as well as the deoxyribose backbone represented by the formation of 8-OH-G. Polyunsaturated fatty acids are extremely sensitive to oxidation with the formation intermediate peroxyl radicals (ROO.) which will finally form the malondialdehyde (MDA) and hydroxynonenal (HNE) as the major toxic end products of lipid peroxidation. Hydroxyl radicals can also react with the residues of proteins, in particular
Pathology of Aging Skin
cystine and methionine to form mixed disulphides. The concentration of carbonyl group and advanced glycation end products (AGEs) serve as a good measure of ROS-mediated protein oxidation. Recent studies have shown that oxidized proteins may lose their structure and aggregate to form protein complexes. Since the antioxidant capacity of tissues decreases during aging, the accumulation of oxidative damage to cells resulting from aerobic metabolism is the molecular basis for the free radical theory of aging process. This has been supported by experimental data showing an accumulation of oxidative damage such as 8-oxo-20 -deoxyguanosine (8-oxodG) residues from DNA, AGEs from proteins, and hydroperoxides and thiobarbituric acid-reacting substances (TBARS) from lipids in the tissues of aged animals [73–78]. Since skin is continuously exposed to oxidative stress from environmental factors and endogenous aerobic metabolism, the damage likely accumulates. It has been found that, in aged rat skin, the oxidized lipid phosphatidylcholine hydroperoxide (PCOOH) increases form 3.46 1.02 mmol/PC mol at 6 months to 7.14 1.63 mmol/PC mol at 24 months. The TBARS content increases from 4.71 1.53 nmol/mg protein at 6 months to 11.10 2.05 nmol/mg protein at 30 months. The free 7-hydroperoxycholesterol (ChOOH) content also increased form 22.83 3.97 at 6 month to 42.58 16.59 mmol/free Ch mol at 24 months. The oxidized DNA in rat skin also increase gradually with age and reach the level 2.04 0.27 8-oxodG/105 dG at 30 months compared to 1.67 0.16 8-oxodG/105 dG at 6 months of age. Although the skin possesses an efficient anti-oxidant activities the increased ROS products in aged skin suggests a chronic accumulation effects during the life time [79–81]. The mitochondria are the primary site for aerobic metabolism. Normal leakage of electron from the electron transport chain makes the mitochondria mtDNA as the primary target of radical oxygen due to the absence of repairing mechanisms within mitochondria. The accumulated mtDNA damage over time will shut down mitochondria function and promote the cell aging and apoptosis [82–85]. In addition, ROS can be produced from a wide range of normal cellular activities including lipoxygenase, COX, plasma membrane NADPH oxidase, NADH dehydrogenase, cytochrome P450, cytochrome b5, microsomal electron transport as well as peroxisome and xanthine oxidase. In addition to already mentioned antioxidant systems, cells also possess an anti-oxidative protein named thioredoxin, which was recently found to play an important role to maintain the ROS homeostasis.
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Experimental evidence showing a decreased level of antioxidant potential in aging cells suggested the redox imbalance at old age. The increased ROS is associated with elevation of MAPKs including ERK, p38 and JNK which in turn will trigger on the NFkB and its downstream pro-inflammatory chemokines and cytokines induction including IL1-b, IL-6, TNF-a, COX2 as well as the VCAM-1 and ICAM-1. So the imbalanced redox might link to its potential inflammatory responses found in aged skin [82, 86, 87].
Conclusion Without question, the skin is a magnificent organ and survival is not possible without it. It is the interface between external and internal worlds, which is nothing short of miraculous. Its beauty is the source of admiration. The primary function of the skin is to serve as a barrier. To this end, the structure of the epidermis is highly regulated in coordinated, dynamic balance between proliferation, differentiation and desquamation. An acidic pH is required to maintain the barrier function by providing an optimal environment for the function of enzymes involved in lipid production/processing, water content and cell–cell conjunction. In addition to its barrier function, the skin also has a role in immunosurveillance, metabolize and sensory reception/transmission. From birth, the skin, like every other organ, begins the journey of aging. The environmental insults which the skin protects against, exact a heavy toll over years. As well, changes in the internal structure, leading to change in the internal milieu further contribute to the decline in structure and function of the skin. It is quite probable that oxidative damage is the singular most damaging event in skin leading to DNA damage, telomere shortening, protein glycosylation and collegen/elastin degradation among several deleterious events. Maintenance of healthy skin throughout life includes moisturization and protection against environmental insults.
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9 Pathomechanisms of Endogenously Aged Skin Evgenia Makrantonaki . Christos C. Zouboulis
Introduction There has been an unprecedented rapid expansion of the population of elderly people both in the developed and developing world [1]. Since 1840, life expectancy has increased at a rate of about 3 months/year [2], whereas the total worldwide aged population is expected to rise from 605 million in 2000 to 1.2 billion in 2025 and to nearly two billion in 2050 [3]. These demographic shifts mandate a better understanding of the aging process and better management strategies for age-associated diseases. Like all other organs, skin suffers progressive morphologic and physiologic decrement with increasing age and provides the first obvious evidence of the aging process. Skin aging can be classified into light-induced aging (photoaging, exogenous aging) and endogenous aging. The latter occurs in nonexposed areas, which are not in direct contact with environmental factors such as ultraviolet (UV) and infrared (IR) irradiation (e.g., the inner side of the upper arm) [4], and is mainly attributed to genetic factors and alterations of the endocrine environment. In contrast to photoaging, endogenously aged skin reflects degradation processes of the entire organism.
Pathomechanisms of Endogenously Aged Skin With advancing age the most pronounced changes in endogenously aged skin occur within the epidermis and affect mostly the basal cell layer. As a result, sun-protected aged skin appears thin, finely wrinkled, and dry (reviewed in > Table 9.1) [4]. Although the fundamental mechanisms are still poorly understood, a growing body of evidence points toward the involvement of multiple pathways in the generation of aged skin. Several theories have been proposed including the theory of cellular senescence [5], decrease in cellular DNA repair capacity and loss of telomeres [6–9], point mutations of extranuclear mitochondrial DNA (mtDNA) [10], oxidative stress [11], increased frequency of chromosomal abnormalities [12, 13],
and gene mutations. In addition, hormones have also been shown to play a distinct role.
Cellular Senescence The theory of cellular senescence describes the observed loss of the cell’s proliferative potential after a limited number of cell divisions [5]. According to this theory, cells possess a ‘‘biological clock,’’ which signals the end of their replicative life span, and as a consequence, they cannot be stimulated to enter the S1 phase by physiological mitogens, arresting at the G1 phase. This process can be partly explained by the selective repression of growth regulatory genes. Studies on keratinocytes [14], fibroblasts [15], and melanocytes [16] have revealed that they all show an age-associated decrease in cumulative population doublings. Fibroblasts, for instance, taken from a normal human tissue go through only about 25–50 population doublings when cultured in a standard mitogenic medium. Towards the end of this time, proliferation slows down and finally stops, and the cells enter a state from which they never recover. The reduction in proliferative capacity of skin-derived cells in culture from old donors and patients with premature aging syndromes and the accumulation in vivo of senescent cells with altered patterns of gene expression also support the theory of cellular senescence.
The Free Radical Theory Oxygen radicals or reactive oxygen species (ROS) are increasingly considered as the major contributors to aging, and the protective mechanism against oxidative stress is observed as an indispensable function [17]. It has been shown that oxygen radical levels rise and antioxidant activity declines with advancing age [18]. Skin possesses many defensive mechanisms in order to reduce the production of ROS from internal sources.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_9, # Springer-Verlag Berlin Heidelberg 2010
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. Table 9.1 Morphological and functional changes in intrinsically aged skin [74] Thinning of epidermis by 10–50%
Increased vulnerability, fragility
Atrophy of the stratum spinosum
Increased vulnerability, fragility
Increased heterogeneity in size of basal cells
Increased vulnerability, fragility
Decreased mitotic activity, increased duration of cell cycle and migration time
Decreased desquamation, delayed wound healing
Slow replacement of lipids
Disturbed barrier function
Flattening of the dermoepidermal junction
Decrease in surface contact area, increased risk of separation by shearing forces
Decrease and heterogeneity of melanocytes
Graying of hair, guttate amelanosis, lentigines
Decrease of Langerhans cells
Diminished cutaneous immune function
Reduction of dermis thickness, decrease of fibroblasts
Reduced strength and resiliency
Atrophy of the extracellular matrix
Reduced strength and resiliency
Reduction and disintegration of collagen and elastic fibers, deposition of exogenous substances (e.g., amyloid P)
Sensitization to deformational forces, fine wrinkle formation
Reduction of cutaneous microvasculature
Reduction of cutaneous vascular responsiveness, disturbed thermoregulation and supply with nutrients
Decrease of skin appendages and their function (e.g., sebaceous Decreased lipid and sweat production, disturbed glands, sweat glands, apocrine glands) re-epithelization of deep cutaneous wounds Thinning of subcutaneous fat
Reduced insulation and energy production
Reduction of nerve endings
Disturbed sensory function
For example, the activity of enzymes that indirectly produce oxygen metabolites can be altered (xanthine oxidase modulation). There is a repair system consisting of enzymes and small molecules, antioxidant enzymes such as catalase and peroxidase, and low-molecular weight antioxidants such as tocopherols, ascorbic acid, NADH, and carnosine, which can donate an electron and then scavenge ROS. Excess ROS production leads to accumulation of cellular damage [19, 20], which includes oxidation of DNA resulting in mutations and oxidation of membrane lipids leading to reduced transport efficiency and altered transmembrane signaling, processes whose consequence is the aging phenotype. A disturbed stress response is also known to be associated with a defect in proteolytic systems such as lysosomal activity and ubiquitine-proteosome pathway in somatic cells [21]. As a consequence, altered proteins cannot be eliminated, and this results in accumulation of misfolded and damaged proteins in the cells. Moreover, cumulative evidence suggests that ROS play a crucial role by participating in multiple MAP kinase pathways, which induce AP-1 and in turn the signal cascade, already mentioned above ( > Fig. 9.1). The free radical theory has also been supported by the fact that strategies that reduce metabolism and the production of ROS, such as dietary caloric restriction (DCR),
can extend the life span of experimental animals. Studies conducted in animal models demonstrated that DCR can retard the aging process by influencing stress response and altering the expression of metabolic and biosynthetic genes [22]. Cancer prevention due to alterations of hormone metabolism, hormone-related cellular signaling, oxidation status, DNA repair, and apoptosis has been also associated with DCR [23, 24]. In skin tissues of mice with DCR weight control, a palette of genes showed a differential expression when compared to mice receiving normal diet [24]. DCR could show profound inhibitory impact on the expression of genes relevant to cancer risks (e.g., neuroblastoma ras oncogene, neuroblastoma mycrelated oncogene 1, Rab40c, myeloblastosis oncogene-like 2, lung carcinoma myc-related oncogene 1, myeloblastosis oncogene, RAB5B, RAP2B, RAB34).
The Telomere Hypothesis The telomere hypothesis of cellular aging [25] proposes that loss of telomeres due to incomplete DNA replication and absence of telomerase provides a mitotic clock that signals cycle exit, limiting the replicative capacity of the somatic cell [9]. Human telomeres consist of repeats of
Pathomechanisms of Endogenously Aged Skin
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. Figure 9.1 A schematic overview of major biochemical changes and signaling pathways involved in the generation of endogenously aged skin. In aged skin, mitogen-activated protein (MAP) kinase signal transduction pathways play an important role in regulating a variety of cellular functions. Downstream effectors of the MAP kinases include several transcription factors including the c-Jun and c-Fos, which heterodimerize in order to form the activator protein 1 (AP-1) complex. AP-1 is a key regulator of skin aging, as it induces the expression of the MMP family and has been shown to inhibit type I procollagen gene expression through interference with TGF-b signaling pathway. It has been postulated that MAP kinases may be activated by excess production of reactive oxygen species (ROS), which occurs with advanced age and may be superimposed by extrinsic factors (e.g., UV/IR irradiation). Excess ROS production also leads to accumulation of cellular damage, which includes oxidation of DNA resulting in mutations, oxidation of proteins leading to reduced function, and oxidation of membrane lipids resulting in reduced transport efficiency and altered transmembrane signaling. NF-kB: nuclear factor-kappa B; TGF-b: transforming growth factor-b; IL-1: interleukin-1; IL-6: interleukin-6; IL-8: interleukin-8
the sequence TTAGGG/CCCTAA at chromosome end, which are not replicated in the same manner as the rest of the genome but instead are synthesized by the enzyme telomerase [9, 26, 27]. By mechanisms that remain unclear, telomerase also promotes the formation of protein cap structures that protect the chromosome ends. Telomerase is active in germline cells and in humans, and telomeres in these cells are maintained at about 15 kilobase pairs (kbp). In contrast, telomerase is not expressed in most human somatic cells like skin cells [7, 28]. As a result, their telomeres become 50–100 nucleotides shorter with every cell division, and their protective protein caps progressively deteriorate. Eventually, after many cell generations, DNA damage occurs at chromosome ends. The damage activates a p53-dependent cell-cycle arrest that
resembles the arrest caused by other types of DNA damage. The lack of telomerase in most somatic cells has been proposed to help protect humans from the potentially damaging effects of runaway cell proliferation, as occurs in cancer. Telomere loss is thought to control entry into senescence [7, 29, 30].
Genes and Mutations The mechanisms which seem to be associated with aging are complex [31]. Recent studies on models such as the yeast Saccharomyces cerevisiae [32], the nematode Caenorhabditis elegans [33], the fly Drosophila melanogaster
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[34–36], the mouse Mus musculus [37], and humans [38] show that single gene mutations can contribute to the initiation of aging and induce premature aging syndromes. However, there are no special genes that can cause agingassociated damages. The manifestation of aging is mostly due to the failure of maintenance and repair mechanisms [39, 40]. Studies on human keratinocytes have demonstrated altered expression of growth-regulating molecules with age; there is an increase of the baseline expression of the differentiation-associated genes like SPR2 and interleukin 1 receptor antagonist [41] and EGF binding and receptor phosphorylation is reduced and thought to be the result of age-related changes in a critical downstream signaling element [42]. In senescent fibroblasts, genes like the c-fos protooncogene [43], the helix-loop-helix Id-1 and Id-2 genes [44], and components of the E2F transcription factor [45] have been shown to be downregulated, and negative growth regulators are overexpressed including the p21 and p16 inhibitors of cyclin-dependent protein kinases [46]. Other changes seen in senescent skin fibroblasts include increased expression of IL-1 and of the EGF-like cytokine heregulin that modulates the growth and differentiation [47]. Moreover, elastin gene expression is markedly reduced after the age of 40–50, as determined by mRNA steady state levels [48]. Furthermore, recent studies indicate that endogenous and exogenous aging may share some fundamental pathways, and may have some common mediators. Photoaging is thought to be the superposition of UV irradiation from the sun on intrinsic aging [49]. Some of the similarities are changes in the MAP kinase signaling pathways, like decreases in ERK-dependent MAP kinase activity and increases in stress-activated JNK and p38 kinase [50], which result in reduced cell proliferation, differentiation, and cell survival [51], and enhanced growth arrest, apoptosis, and stress-related responses [51, 52]. As a consequence of the stressactivated MAP kinase pathways, the expression of c-jun and c-Jun N terminal kinase – an upstream activator of c-jun, is elevated in aged compared with young skin [50]. As c-jun is a constituent of the transcription factor AP-1, AP-1 is also elevated and subsequently the AP-1 regulated connective tissue-degrading enzymes MMP-1 (interstitial collagenase), MMP-3 (stromelysin 1), and MMP-9 (gelatinase B). In parallel, there is an observed reduction in the expression of tissue inhibitors of metalloproteinases [53, 54]. Another common feature is the increased insoluble degraded collagen and the reduction of types I
and III procollagen synthesis, which may result from the impaired TGFb signaling pathway [53, 55]. In recent studies, researchers have been focusing on gene mutations accompanying known progeroid syndromes, e.g., Hutchinson–Gilford progeria, Werner’s syndrome (WS), Rothmund–Thomson syndrome, Cockayne syndrome, Ataxia teleangiectasia, and Down syndrome. The most common skin disorders of these syndromes, which are characterized by an acceleration of the aging phenotype, are alopecia, skin atrophy and sclerosis, teleangiectasia, poikiloderma, thinning and graying of hair, and several malignancies. Most of these syndromes are inherited in an autosomal recessive way and mostly display defects in DNA replication, recombination, repair, and transcription. Expression gene patterns of skin cells derived from Werner patients [56], old and young donors showed that 91% of the analyzed genes had similar expression changes in WS and in normal aging implying transcription alterations common to WS and normal aging represent general events in the aging process. Ly et al. measured mRNA levels in fibroblasts isolated from young, middle-aged, and elderly patients with progeria and found chromosomal pathologies that lead to misregulation of key structural, signaling, and metabolic genes associated with the aging phenotype [13]. Further studies conducted to investigate changes in gene expression during skin aging have been performed on naturally aged human foreskin obtained from children and elderly males. Some of the mechanisms proposed to be involved in the induction of aging comprise disturbed lipid metabolism, altered insulin and STAT3 signaling, upregulation of apoptotic genes partly due to the deregulation of FOXO1, dowregulation of members of the jun and fos family, differential expression of cytoskeletal proteins (e.g., keratin 2A, 6A, and 16A), extracellular matrix components (e.g., PI3, S100A2, A7, A9, SPRR2B), and proteins involved in cell-cycle control (e.g., CDKs, GOS2) [57].
The Mitochondrial DNA Theory Genetic damage and instability outside the nuclear genome has been also suggested to contribute to aging [58]. The mtDNA synthesis takes place near the inner mitochondrial membrane, which is the site of formation of ROS, and the fact that mtDNA lacks excision and recombination repair has made many investigators believe that cumulative damage of the mtDNA may play a key role in the pathogenesis of the aging phenotype [10, 11].
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Examination of human fibroblast mtDNA in aged individuals revealed point mutations at specific positions in the control region for replication. Notably, a T414G transversion was found in a significantly higher proportion of persons older than 65 years when compared with younger persons [11].
Hormone Decline and Skin Aging One of the further factors that may play a predominant role in the initiation of skin aging is the physiological hormone decline occurring with age. Over time important circulating hormones decline due to a reduced secretion of the pituitary, adrenal glands and the gonads, or due to an intercurrent disease. Among them, growth factors (i.e., growth hormone [GH] and insulin-like growth factor-I [IGF-I]) and sex steroids (e.g., androgens and estrogens) show significant changes in their blood levels. In animal models, such as in organisms as diverse as the nematode C. elegans, the fly D. melanogaster, and the mouse M. musculus, the importance of hormonal signals on the aging phenotype has already been documented. Suppression of hormones such as insulin-like peptides, growth hormone (GH), and sterols [59], or their receptors can increase life span and delay age-dependent functional decline. Conboy et al. [60] showed that the age-related decline of progenitor cell activity of mice could be reversed by exposure to young serum and that the cells could retain much of their intrinsic proliferative potential even when old, underlining the great importance of the systemic environment. In an in vitro model of human hormonal aging, human skin cells cultured under hormone-substituted conditions showed altered lipid synthesis and metabolism and affected expression of genes being involved in biological processes, such as DNA repair and stability, mitochondrial function, oxidative stress, cell cycle and apoptosis, ubiquitin-induced proteolysis, and transcriptional regulation indicating that these processes may be hormone-dependent [61]. These studies illustrate the importance of the hormone environment for deterioration of the human organism and the aging process. The growth hormone (GH)/insulin-like growth factorI (IGF-I) axis is considered to be one of the most important signaling pathways involved in aging. Serum levels of IGF-I have been reported to increase from birth to puberty, followed by a slow decline through adulthood. This reduction has been correlated with the progressive decline of GH with advancing age [62]. Patients with isolated GH
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deficiency (IGHD), multiple pituitary hormone deficiency (MPHD) including GH, as well as primary IGF-I deficiency (GH resistance, Laron syndrome) present signs of early skin aging such as dry, thin, and wrinkled skin. Other resulting characteristics of GH/IGF-I deficiency are obesity, hyperglycemia, reduced body lean mass, osteopenia, lowered venous access, hypercholesterolemia, cardiovascular diseases and, subsequently, premature mortality [63–65]. Treatment of normal elderly males with GH resulted in amelioration and reverse of the aging signs and symptoms [66]. However, recent reports of an association of GH substitution and increased risk of prostate, lung, colon, breast cancer, as well as a possible decrease of insulin insensitivity all make further investigations necessary regarding safety and efficacy of GH substitution in the aging population [67]. On the other hand, menopause, which is characterized by a rapid decline of sex steroids, has been associated with a worsening of skin structure and functions, which can be at least partially repaired by hormone replacement therapy or local estrogen treatment [68]. Improvement of epidermal skin moisture, elasticity and skin thickness [69], enhanced production of surface lipids [70], reduction of wrinkle depth, restoration of collagen fibers [71], and increase of the collagen III/I ratio [72] have all been reported under hormone replacement therapy. In vitro test that studied the effects of GH, IGF-I androgens, and estrogens at age-specific levels on human skin cells have been documented. IGF-I was shown to play an important role in the regulation of the lipid synthesis in human sebocytes, while 17b-estradiol showed no significant effects on the biological activity of the cells. Dermal fibroblasts showed to be more susceptible to 17b-estradiol treatment, while IGF-I could significantly stimulate fibroblast proliferation. Furthermore, an interplay between the 17b-estradiol and IGF-I signaling pathways was documented in both cell types [73]. These results indicate the importance of IGF-I in the reduction of skin surface lipids and thickness with advanced age.
Conclusion In summary, several factors may contribute to endogenous skin aging underlining the complexity of this phenomenon. Amongst these are excess production of reactive oxygen species and impaired scavenge mechanisms, increased frequency of chromosomal abnormalities, telomere loss and point mutations of extranuclear mitochondrial DNA due to reduced DNA repair capacity.
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In addition, several genes and their mutations have been correlated with the aging phenotype. Hormones and their physiological decline with time also play a distinct role as shown in several in vitro and in vivo studies. Like the entire organism, skin follows the pathway of aging with time. In addition to internal factors, several environmental factors contribute to this process and sometimes accelerate the onset of aging. Skin functions deteriorate and this results in the development of a palette of diseases, which may jeopardise life quality. Awareness of the pathophysiology of skin aging as well as of preventive measures to avoid skin damage is the first step for successful, healthy aging.
Cross-references > Pathomechanisms
of Photoaged Skin
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10 Pathomechanisms of Photoaged Skin Jean Krutmann
Introduction Among all environmental factors, solar UV radiation is the most important in premature skin aging, a process accordingly termed photoaging. Over recent years, substantial progress has been made in elucidating the underlying molecular mechanisms. From these studies, it is now clear that both UVB (290–320 nm) and UVA (320– 400 nm) radiations contribute to photoaging. UVinduced alterations at the level of the dermis are best studied and appear to be largely responsible for the phenotype of photoaged skin. It is also generally agreed that UVB acts preferentially on the epidermis where it not only damages DNA in keratinocytes and melanocytes, but also causes the production of soluble factors including proteolytic enzymes, which in a second step affect the dermis; in contrast, UVA radiation penetrates far more deeply on average and hence exerts direct effects on both the epidermal and the dermal compartments (> Fig. 10.1). UVA is also 10–100 times more abundant in sunlight than UVB, depending on the season and time of the day. Therefore, it has been proposed that, although UVA photons are individually far less biologically active than UVB photons, UVA radiation may be at least as important as UVB radiation in the pathogenesis of photoaging [1]. The exact mechanisms by which UV radiation causes premature skin aging is not yet clear, but a number of molecular pathways explaining one or more of the key features of photoaged skin have been described. Some of these models are based on irradiation protocols, which use single or few UV exposures, whereas others take into account the fact that photoaging results from chronic UV damage, and as a consequence employ chronic repetitive irradiation protocols. Still others rely on largely theoretical constructs rather than experimental observations.
Mechanisms of Photoaging All organ systems are affected by aging processes, many in organ-specific ways; however, aging uniformly has the effect of reducing maximal function and reserve
capacity, as well as the ability to compensate for injury and a hostile environment. Ultimately, such losses are incompatible with life. Of interest, most, if not all, age-accelerating environmental factors damage DNA either directly or indirectly, often through oxidation. Furthermore, the rate of aging in various species correlates [2] inversely with the rate and fidelity of DNA repair [3], and most progeroid syndromes for which the genetic lesion has been identified have impaired DNA replication and/or DNA damage responses. In combination with the fact that cumulative DNA damage accompanies chronological aging [4], these observations suggest that both the indisputable heritable and the environmental components of aging result in large part from changing DNA status during the individual’s lifetime. The next section develops this intellectual framework and relates it to the phenomenon of skin aging, and particularly photoaging, by focusing on mtDNA [5, 1]. The subsequent sections provide detailed information now available with regard to specific aging targets and signaling pathways responsible for photoaging-associated morphological and functional changes in the skin. These include UV-induced alterations of connective tissue components, vascularization patterns, inflammatory cells, and protein oxidation. Finally, a unifying concept is presented that reconciles with the most recent findings in an attempt to provide a novel and comprehensive model to explain photoaging and provide a framework for future investigations.
Mitochondrial DNA Mutations and Photoaging Mitochondria are organelles whose main function is to generate energy for the cell. This is achieved by a multistep process called oxidative phosphorylation or electron transport chain. Located at the inner mitochondrial membrane are five multiprotein complexes that generate an electrochemical proton gradient used in the last step of the process to turn ADP and organophosphate into ATP. This process is not completely error-free and ultimately
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_10, # Springer-Verlag Berlin Heidelberg 2010
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. Figure 10.1 Wavelength-dependent penetration of UV radiation into human skin
leads to the generation of reactive oxygen species (ROS), making the mitochondrion the site of the highest ROS turnover in the cell. In close proximity to this site lies the mitochondrion’s own genomic material, the mtDNA. Human mtDNA is a 16,559-bp long circular doublestranded molecule of which four to ten copies exist per cell. Since mitochondria do not exhibit any repair mechanism to remove bulky DNA lesions, although they do exhibit a base excision repair mechanism and repair mechanisms against oxidative damage, the mutation frequency of mtDNA is approximately 50-fold higher than that of nuclear DNA. Mutations of mtDNA have been found to play a causative role in degenerative diseases such as Alzheimer’s disease, chronic progressive external ophthalmoplegia, and Kearns–Sayre syndrome (KSS) [6]. In addition to degenerative diseases, mutations of mtDNA may play a causative role in the normal aging process with an accumulation of mtDNA mutations accompanied by a decline of mitochondrial functions [7]. Recent evidence indicates that mtDNA mutations are also involved in the process of photoaging [1]. Photoaged skin is characterized by increased mutations of the mitochondrial genome [8, 9, 10]. Intraindividual comparison studies have revealed that the so-called common deletion, a 4,977-bp deletion of mtDNA, is
increased up to tenfold in photoaged skin as compared with sun-protected skin of the same individuals. The amount of the common deletion in human skin does not correlate with chronological aging [11], and it has therefore been proposed that mtDNA mutations such as the common deletion represent molecular markers for photoaging. In support of this concept it has been shown that repetitive, sublethal exposure to UVA radiation at doses that may be acquired during a regular summer holiday induces mutations of mtDNA in cultured primary human dermal fibroblasts in a singlet oxygendependent fashion [3]. Even more importantly, in vivo studies have revealed that repetitive exposure three times daily of previously unirradiated buttock skin for a total of 2 weeks to physiological doses of UVA radiation leads to an approximately 40% increase in the levels of the common deletion in the dermal, but not the epidermal, compartment of irradiated skin [12]. Furthermore, it has been shown that, once induced, these mutations persist for at least 16 months in UV-exposed skin. Interestingly, in a number of individuals, the levels of the common deletion in irradiated skin continued to increase with a magnitude up to 32-fold. It has been postulated for the normal aging process as well as for photoaging that the induction of ROS generates mtDNA mutations, in turn leading to a defective respiratory chain and, in a vicious cycle, inducing even more ROS and subsequently allowing mtDNA mutagenesis independent of the inducing agent [13]. It is the characteristic of vicious cycles that they evolve at everincreasing speeds. Thus, the increase of the common deletion up to levels of 32-fold, independent of UV exposure may represent the first in vivo evidence for the presence of such a vicious cycle in general and in human skin in particular. The mechanisms by which generation of mtDNA mutations by UVA exposure translates into the morphological alterations observed in photoaging human skin are currently being unravelled. In general, a cause-effect relationship between premature aging and mtDNA mutagenesis is strongly suggested by studies employing homozygous knock-in mice that express a proofreading-deficient version of PolgA, the nucleus-encoded subunit of mtDNA polymerase [14]. As expected, these mice develop an mtDNA mutator phenotype with increased amounts of deleted mtDNA. This increase in somatic mtDNA mutations has been found to be associated with a reduced life span and premature onset of aging-related phenotypes such as weight loss, reduced subcutaneous fat, alopecia, kyphosis, osteoporosis, anemia, reduced fertility, and heart enlargement.
Pathomechanisms of Photoaged Skin
In addition, recent studies have demonstrated that UVA radiation-induced mtDNA mutagenesis is of functional relevance in primary human dermal fibroblasts and apparently has molecular consequences suggestive of a causative role for mtDNA mutations in photoaging of human skin as well [15]. Accordingly, induction of the common deletion in human skin fibroblasts is paralleled by a measurable decrease of oxygen consumption, mitochondrial membrane potential, and ATP content, as well as an increase of MMP-1, while tissue-specific inhibitors of MMPs (TIMPs) remain unaltered, an imbalance that is known to be involved in photoaging of human skin (see below). These observations suggest a link not only between mutations of mtDNA and cellular energy metabolism, but also between mtDNA mutagenesis, energy metabolism, and a fibroblast gene expression profile that would functionally correlate with increased matrix degradation and thus premature skin aging. In order to provide further evidence for a role of the energy metabolism in mtDNA mutagenesis and the development of this ‘‘photoaging phenotype,’’ the effect of creatine was studied in these cells. This applied the hypothesis that generation of phosphocreatine, and consequently ATP, is facilitated if creatine is abundant in cells. This would allow easier binding of existing energy-rich phosphates to the energy precursor creatine. Indeed, experimental supplementation of normal human fibroblasts with creatine normalizes mitochondrial mutagenesis as well as the functional parameters, oxygen consumption, and MMP-1, while an inhibitor of creatine uptake abrogates this effect [15] (> Fig. 10.2). A second line of evidence for cause-effect relationship between a disturbance of mtDNA integrity and skin aging is provided by a very recent study, in which a phenocopy of cells bearing large-scale deletions of mtDNA was generated by gradually depleting the mtDNA from unirradiated human skin fibroblasts [16]. Gradual depletion of mtDNA caused a gene expression profile, which was a reminiscent of that observed in photoaged skin. Accordingly, in these cells an increased expression of MMP-1 without a concomitant change in tissue inhibitor metalloproteinase-1 as well as a decreased expression of collagen type 1 alpha-1, that is, a gene involved in collagen de novo synthesis, was observed. This altered gene expression resulted from intracellular, mitochondria-derived oxidative stress. These results support the concept that disruption of mt integrity, for example, by UV-induced mtDNA mutagenesis, is of pathogenetic relevance for photoaging of human skin. Finally, a third line of evidence for a pathogenetic role of mtDNA mutations in photoaging is provided by
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. Figure 10.2 The defective powerhouse model of (photo)aging of the skin
human in vitro studies employing skin fibroblasts from Kearns–Sayre syndrome (KSS) patients, which constitutionally carry large amounts of the UV-inducible common deletion. Accordingly, human dermal skin equivalent models, which were engineered using KSS skin fibroblasts, developed multiple features of photoaged skin including MMP-1 upregulation and increased collagen breakdown even in the absence of any UV exposure ([17] and Krutmann et al., unpublished observation).
Connective-Tissue Alterations in Photoaging: The Role of Matrix Metalloproteinases and Collagen Synthesis Photoaged skin is characterized by alterations to the dermal connective tissue. The extracellular matrix in the dermis mainly consists of type I and type III collagen, elastin, proteoglycans, and fibronectin. In particular,
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collagen fibrils are important for the strength and resilience of skin, and alterations in their number and structure are thought to be responsible for wrinkle formation. In photoaged skin, collagen fibrils are disorganized and abnormal elastin-containing material accumulates [18]. Biochemical studies have revealed that in photoaged skin levels of types I and III collagen precursors and crosslinks are reduced, whereas elastin levels are increased [19, 20]. How does UV radiation cause these alterations? In principle it is reasonable to assume that UV radiation leads to an enhanced and accelerated degradation and/or a decreased synthesis of collagen fibers, and current knowledge indicates that both mechanisms may be involved. A large number of studies unambiguously demonstrate that the induction of matrix metalloproteinases (MMPs) plays a major role in the pathogenesis of photoaging. As indicated by their name, these zinc-dependent endopeptidases show proteolytic activity in their ability to degrade matrix proteins such as collagen and elastin. Each MMP degrades different dermal matrix proteins; for example, MMP-1 cleaves collagen types I, II, and III, whereas MMP-9, which is also called gelatinase, degrades collagen types IV and V and gelatin. Under basal conditions, MMPs are part of a coordinated network and are precisely regulated by their endogenous inhibitors, i.e., TIMPs, which specifically inactivate certain MMPs. An imbalance between activation of MMPs and their respective TIMPs could lead to excessive proteolysis. It is now very well established that UV radiation induces MMPs without affecting the expression or activity of TIMPs [21, 22]. These MMPs can be induced by both UVB and UVA radiations, but the underlying photobiological and molecular mechanisms differ depending on the type of irradiation. In a very simplified scheme, UVA radiation would mostly act indirectly through the generation of ROS, in particular, singlet oxygen, which can subsequently exert a multitude of effects such as lipid peroxidation, activation of transcription factors, and generation of DNA strand breaks [22]. While UVB radiationinduced MMP induction has been shown to involve the generation of ROS as well [23], the main mechanism of action of UVB is by direct interaction with DNA via the induction of DNA damage. Recent studies have indeed provided evidence that enhanced repair of UVB-induced cyclobutane pyrimidine dimers in the DNA of epidermal keratinocytes through topical application of liposomally encapsulated DNA repair enzymes on UVB-irradiated human skin prevents UVB radiation-induced epidermal MMP expression. [24].
The activity of MMPs is tightly regulated by transcriptional regulation and elegant in vivo studies by Fisher et al. have demonstrated that exposure of human skin to UVB radiation leads to the activation of the respective transcription factors [25]. Accordingly, UV exposure of human skin not only leads to the induction of MMPs within hours after irradiation, but already within minutes, transcription factors AP-1 and NF-kB, which are known stimulatory factors of MMP genes, are induced. These effects can be observed at low UVB dose levels, because transcription factor activation and MMP-1 induction can be achieved by exposing human skin to one tenth of the dose necessary for skin reddening (0.1 minimal erythema dose). Subsequent work by the same group clarified the major components of the molecular pathway by which UVB exposure leads to the degradation of matrix proteins in human skin. Low-dose UVB irradiation induces a signaling cascade, which involves upregulation of epidermal growth factor receptors (EGFR), the GTP-binding regulatory protein p21Ras, extracellular signal-regulated kinase (ERK), c-jun aminoterminal kinase (JNK), and p38. Elevated c-jun together with constitutively expressed c-fos increases activation of AP-1. Identification of this UVB-induced signaling pathway not only unravels the complexity of the molecular basis, which underlies UVB radiation-induced gene expression in human skin, but also provides a rationale for the efficacy of tretinoin (alltrans-retinoic acid) in the treatment of photoaged skin. Accordingly, topical pretreatment with tretinoin inhibits the induction and activity of MMPs in UVB-irradiated skin through prevention of AP-1 activation. In addition to destruction of existing collagen through activation of MMPs, failure to replace damaged collagen is thought to contribute to photoaging as well. Accordingly, in chronically photodamaged skin, collagen synthesis is down regulated as compared to sun-protected skin [26]. The mechanism by which UV radiation interferes with collagen synthesis is not yet known, but a recent study has provided evidence that fibroblasts in severely (photo) damaged skin have less interaction with intact collagen and are thus exposed to less mechanical tension, and it has been proposed that this situation might lead to decreased collagen synthesis [27].
UV-Induced Modulation of Vascularization There is increasing evidence that cutaneous blood vessels may play a role in the pathogenesis of photoaging. Photoaged skin shows vascular damage intrinsically aged skin
Pathomechanisms of Photoaged Skin
does not. In mildly photodamaged skin, there is venular wall thickening, while in severely damaged skin the vessel walls are thinned and the supporting perivascular veil cells are reduced in number [28]. The number of vascular cross-sections is reduced [29] and there are local dilations, corresponding to clinical telangiectases. Overall, there is a marked change in the horizontal vascularization pattern with dilated and distorted vessels. Studies in humans and in the hairless skh-1 mouse model for skin aging have demonstrated that acute and chronic UVB irradiation greatly increases skin vascularization [30, 31]. The formation of blood vessels from preexisting vessels is tightly controlled by a number of angiogenic factors and factors that inhibit angiogenesis. These growth factors include basic fibroblast growth factor, interleukin-8, tumor growth factor-beta, platelet-derived growth factor, and vascular endothelial growth factor (VEGF). VEGF appears to be involved in chronic UVB damage because UVB radiation-induced dermal angiogenesis in Skh-1 mice is associated with increased VEGF expression in the hyperplastic epidermis of these animals [31]. Even more importantly, targeted overexpression of the angiogenesis inhibitor thrombospondin-1 not only prevents UVB radiation-induced skin vascularization and endothelial cell proliferation, but also significantly reduces dermal photodamage and wrinkle formation. These studies suggest that UVB radiation-induced angiogenesis plays a direct biological role in photoaging.
Photoaging as a Chronic Inflammatory Process In contrast to intrinsically aged skin, which shows an overall reduction in cell numbers, photoaged skin is characterized by an increase in the number of dermal fibroblasts, which appear hyperplastic, and also by increased numbers of mast cells, histiocytes, and mononuclear cells. The presence of such a dermal infiltrate indicates the possibility that a chronic inflammatory process takes place in photoaged skin, and in order to describe this situation the terms heliodermatitis and dermatoheliosis have been coined [32]. More recent studies have shown that increased numbers of CD4 + T cells are present in the dermis, whereas intraepidermally, infiltrates of indeterminate cells and a concomitant reduction in the number of epidermal Langerhans cells have been described [33]. It is currently not known whether the presence of inflammatory cells represents an epiphenomenon or whether these cells play a causative role in the pathogenesis of photoaging, e.g., through the production of soluble mediators,
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which could affect the production and/or degradation of extracellular matrix proteins.
Protein Oxidation and Photoaging The aging process is accompanied by enhanced oxidative damage. All cellular components including proteins are affected by oxidation [34]. Protein carbonyls may be formed either by oxidative cleavage of proteins or by direct oxidation of lysine, arginine, proline, and threonine residues. In addition, carbonyl groups may be introduced into proteins by reaction with aldehydes produced during lipid peroxidation or with reactive carbonyl derivatives generated as a consequence of the reaction with reducing sugars or their oxidation products with lysine residues of proteins. Within the cell, the proteasome is responsible for the degradation of oxidized proteins. During the aging process, this function of the proteasome is diminished and oxidized proteins accumulate. In addition, lipofuscin, a highly cross-linked and modified protein aggregate, is formed. This aggregate accumulates within cells and is able to inhibit the proteasome. These alterations mainly occur within the cytoplasm and lipofuscin does not accumulate in the nucleus. In biopsies from individuals with histologically confirmed solar elastosis, an accumulation of oxidatively modified proteins was found specifically within the upper dermis [35]. Protein oxidation in photoaged skin is most likely due to UV irradiation because repetitive exposure of human buttock skin over 10 days to increasing UV doses as well as in vitro irradiation of cultured dermal fibroblasts with UVB or UVA have been shown to cause protein oxidation. The functional relevance of increased protein oxidation in UV-irradiated dermal fibroblasts, in particular with regard to the pathogenesis of photoaging, is currently not known. Very recent studies, however, indicate that increased protein oxidation, which may result from a single exposure of cultured human fibroblasts to UVA radiation, inhibits proteasomal functions and thereby affects intracellular signaling pathways, which are involved in MMP-1 expression (Krutmann J et al., unpublished observation).
Conclusion and Towards a Unifying Concept From the above observations, it is evident that major progress has been made recently in identifying molecular
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mechanisms involved in photoaging. In this regard, skin has proven to serve as an excellent model organ to understand basic mechanisms relevant for extrinsic aging. Despite all these progresses, however, a general, unifying concept linking the different mechanisms and molecular targets described in the previous paragraphs is still lacking. In other words, the critical question to answer is: How do mtDNA mutagenesis, neovascularization, protein oxidation, down regulation of collagen synthesis, and increased expression of matrix metalloproteinases together cause photoaging of human skin? Which of these mechanisms are of primary importance and responsible for inducing others? Are some or all of the above-mentioned characteristics of photoaged skin merely epiphenomena and, if so, to what extent are they causally related to premature skin aging? The current state of knowledge does not allow to answer these questions in a definitive manner. Nevertheless, a hypothesis is proposed, which tries to reconcile with most of the research discussed above in one model, for which the term ‘‘the defective powerhouse model of skin aging’’ has been coined [16, 36]. Specifically, the persistence of UV radiation-induced mtDNA mutations and the resulting vicious cycle with further increases in mtDNA mutations lead to a situation resembling a ‘‘defective powerhouse’’ where inadequate energy production leads to chronic oxidative stress. In the dermis, functional consequences of direct DNA damage and aberrant ROS production in human dermal fibroblasts could be (1) an altered gene expression pattern, which would affect neovascularization and collagen metabolism and possibly also the generation of an inflammatory infiltrate, (2) the oxidation of intracellular proteins and inhibition of the proteasome, and (3) possibly also changes in the epidermal compartment such as epidermal atrophy, skin barrier, and dysfunction. As a consequence, measures to prevent, delay, or even reverse photoaging should target the dermal rather than the epidermal compartment of human skin.
Cross-references > Pathomechanisms
of Endogenously Aged Skin
Acknowledgments Part of the work described in this chapter has been supported by the DFG, SFB 728.
References 1. Berneburg M, Plettenberg H, Krutmann J. Photoaging of human skin. Photodermatol Photoimmunol Photomed. 2000;16:239–244. 2. Brash D, Rudolph J, Simon J, Lin A, McKenna G, Baden H, Halperin A, Pontenm JA. Role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc Natl Aacad Sci USA. 1998;88:10124–10128. 3. Hart RW, Setlow RB. Correlation between deoxyribonucleic acid excision-repair and life-span in a number of mammalian species. Proc Natl Acad Sci USA. 1974;71:2169–2173. 4. Vijg J. Somatic mutations and aging: a re-evaluation. Mutat Res. 2000;447:117–135. 5. Berneburg M, Grether-Beck S, Kurten V, Ruzicka T, Briviba K, Sies H, Krutmann J. Singlet oxygen mediates the UVA-induced generation of the photoaging-associated mitochondrial common deletion. J Biol Chem. 1999;274:15345–15349. 6. DiMauro S, Schon EA. Mitochondrial respiratory-chain diseases. N Engl J Med. 2003;348:2656–2668. 7. Wallace DC. Mitochondrial genetics: a paradigm for aging and degenerative diseases? Science. 1992;256:628–632. 8. Berneburg M, Gattermann N, Stege H, Grewe M, Vogelsang K, Ruzicka T, Krutmann J. Chronically ultraviolet-exposed human skin shows a higher mutation frequency of mitochondrial DNA as compared to unexposed skin and the hematopoietic system. Photochem Photobiol. 1997;66:271–275. 9. Birch-Machin MA, Tindall M, Turner R, Haldane F, Rees JL. Mitochondrial DNA deletions in human skin reflect photo- rather than chronologic aging. J Invest Dermatol. 1998;110:149–152. 10. Yang JH, Lee HC, Wei YH. Photoageing-associated mitochondrial DNA length mutations in human skin. Arch Dermatol Res. 1995;287:641–648. 11. Koch H, Wittern KP, Bergemann J. In human keratinocytes the common deletion reflects donor variabilities rather than chronologic aging and can be induced by ultraviolet A irradiation. J Invest Dermatol. 2001;117:892–897. 12. Berneburg M, Plettenberg H, Medve-Konig K, Pfahlberg A, GersBarlag H, Gefeller O, Krutmann J. Induction of the photoagingassociated mitochondrial common deletion in vivo in normal human skin. J Invest Dermatol. 2004;12:1277–1283. 13. Jacobs HT. The mitochondrial theory of aging: dead or alive? Aging Cell. 2003;2:11–17. 14. Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder E, Bohlooly YM, Gidlof S, Oldfors A, Wibom R, Tornell J. Jacobs HT, Larsson NG. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004;429: 417–423. 15. Berneburg M, Gremmel T, Kurten V, Schroeder P, Hertel I, Mikecz AV, Wild S, Chen M, Declercq L, Matsui M, Ruzicka T, Krutmann J. Creatine supplementation normalizes mutagenesis of mitochondrial DNA as well as functional consequences. J Invest Dermatol. 2005;125:213–220. 16. Schroeder P, Gremmel T, Berneburg M, Krutmann J. Partial depletion of mitochondrial DNA from human skin fibroblasts induces a gene expression profile reminiscent of photoaged skin. J Invest Dermatol. 2008;128:2297–2303. 17. Majora M, Wittkampf T, Schuermann B, Grether-Beck S, Schroeder P, Krutmann J. Increased expression of lysyl oxidase and production of reactive oxygen species in fibroblasts with deletions of the
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27. Varani J, Schuger L, Dame MK, Leonhard Ch, Fligiel SEG, Kang S, Fisher GJ, Vorhees JJ. Reduced fibroblast interaction with intact collagen as a mechanism for depressed collagen synthesis in photodamaged skin. J Invest Dermatol. 2004;122: 1471–1479. 28. Braverman M, Fonfrko E. Studies in cutaneous aging: II. The microvasculature. J Invest Dermatol. 1982;73:59–66. 29. Kligman AM. Perspectives and problems in cutaneous gerontology. J Invest Dermatol. 1979;73:39–46. 30. Bielenberg DR, Bucana CD, Sanchez R, Donawho CK, Kripke ML, Fidler IJ. Molecular regulation of UVB-induced angiogenesis. J Invest Dermatol. 1998;111:864–872. 31. Yano K, Ouira H, Detmar M. Targeted over expression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photodamage. J Invest Dermatol. 2002;118: 800–805. 32. Lavker RM, Kligman A. Chronic heliodermatitis: a morphologic evaluation of chronic actinic dermal damage with emphasis on the role of mast cells. J Invest Dermatol. 1988;90:325–330. 33. DeLeo VA, Dawes L, Jackson R. Density of Langerhans cells (LC) in normal versus chronic actinically damaged skin (CADS) of humans. J Invest Dermatol. 1981;76:330–334. 34. Levine RL, Stadtman ER. Oxidative modification of protein during ageing. Exp Gerontol. 2001;36:1495–1502. 35. Sander CS, Chang H, Salzmann S, Muller CS, EkanayakeMudiyanselage S, Elsner P, Thiele JJ. Photoaging is associated with protein oxidation in human skin in vivo. J Invest Dermatol. 2002;118:618–625. 36. Schroeder P, Krutmann J. Role of mitochondria in photoageing of human skin: the defective powerhouse model. J Invest Dermatol Symp P. 2009;in press.
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21 Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin Stacy S. Hawkins . William Shingleton . Jean Adamus . Helen Meldrum
Introduction The outermost layers of the epidermis are critical for providing a barrier against environmental factors, such as damage due to chronic UV irradiation and pollutants. An intact epidermal barrier is critical for preventing moisture loss and maintaining the health, function, and attractive appearance of the skin throughout the aging process [1–3]. Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. Other nuclear hormone receptors include steroid hormones, thyroid hormones, and vitamin D hormones, and there is evidence that, similar to other nuclear receptors, PPARs perform a significant role in skin homeostasis [4]. Like other nuclear receptor superfamily members, PPARs contain both a ligand-binding domain and a DNA-binding domain. On ligand activation, PPARs form a heterodimer with retinoic X receptors (RXRs); the resulting complex then binds to PPAR-response elements (PPREs), leading to either an increase or a decrease in the expression of target genes [4, 5]. Although PPAR target genes have been identified across many clinical applications (e.g., cell proliferation, differentiation, inflammation and angiogenesis, atherosclerosis, lipid and glucose metabolism [4]), the emphasis of this summary will be the role of PPARs in the maintenance and improvement of human epidermal skin conditions.
PPARs: Role in Epidermal Structure and Function PPAR-mediated effects on skin homeostasis as well as disease treatment are now well accepted, and it has been shown that activators of PPARs are important regulators
of epidermal differentiation. Since the potential roles of PPARs were first identified by Issemann and Green in 1990 [6], three isotypes have been identified in the human epidermis to date: PPARa, PPAR b/d, and PPARg [7–9]. Over the past 15 years, PPARs have been studied for many clinical applications in dermatology, such as epidermal permeability barrier development and homeostasis [10, 11], stimulation of epidermal keratinocyte differentiation [5, 12–15], and keratinocyte response to inflammation [16, 17], and for treatment of epidermal disorders characterized by inflammation, keratinocyte hyperproliferation, and abnormal differentiation such as psoriasis [18–20]. Several pharmaceutical PPAR ligands have been developed; however, the endogenous ligands are naturally occurring fatty acids. PPAR lipids have also been shown to increase expression of epidermal differentiation proteins in vitro (e.g., transglutaminase [Tgase], involucrin, profilaggrin) and promote cornified cell envelope (CE) maturation in normal human keratinocytes [5]. PPARa agonists have been shown to increase the synthesis of epidermal lipids critical to maintaining a healthy permeability barrier in a living skin equivalent model [21]. Knockout and mutant mouse models have been used to determine the importance of PPARa in epidermal maturation and homeostasis. Analysis has shown that although the barrier function was normal, the stratum granulosum was thinner, with focal parakeratosis, suggesting impaired keratinocyte differentiation, as reflected by a moderate decrease in differentiation markers [22]. A recent study has demonstrated a role for PPAR agonists in improvement in skin barrier in a newborn mouse model, through increased lipid synthesis and acidification [23]. In skin, induction of PPARg results in skin inflammatory diseases such as atopic and allergic contact dermatitis or psoriasis. Topical PPARg agonists troglitazone and ciglitazone increased differentiation markers and
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_21, # Springer-Verlag Berlin Heidelberg 2010
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promoted epidermal barrier recovery in hairless mice [14], possibly through ERK1/2 and p38 signaling [24]. PPAR b/d is the most abundant of the three isotypes found in skin, yet functionally less defined due to its lack of specific ligands [8, 25]. In keratinocytes, it has been shown to be involved in differentiation, lipid accumulation, directional sensing, polarization and migration [15, 16]. PPARb/d may also play a role in the epidermal barrier formation, as evidenced in the LSE model [21] and also in primary keratinocytes [26]. In barrier-compromised skin such as the axillary area, PPAR ligands have been shown to improve epidermal skin condition, as measured by increased involucrin and filaggrin by immunohistochemistry compared to vehicle control, following a repeat patch test [27]. More recently, PPAR lipids have been shown to significantly improve the photoaged appearance after long-term product application [28, 29].
PPARs: Role in Inflammation and Skin Aging Inflammation is a contributing factor in many aging processes in the human body, from intrinsic aging to the pathogenesis of age-related diseases such as atherosclerosis, Alzheimer’s disease, and arthritis. There is a wealth of information in the literature documenting the anti-inflammatory role of PPARs in a variety of animal models, cell types, human tissues and diseases [30–35]. Therefore, this section of the review focuses on the skin-specific influences of PPARs on inflammation. In the field of dermatology, inflammation or irritation is associated with many diseases, and activation of PPARs has been shown to have beneficial effects on a number of skin conditions [17, 34, 36–38]. Marketed pharmaceuticals for the treatment of inflammatory skin disorders include agonists to PPARa and PPARg. The mode of action of these drugs is, in part, through reduction of inflammation. Concomitant with this appears to be a normalization of epidermal turnover [39]. The expression of PPARa and PPARg in psoriatic skin is unchanged or even reduced; however, PPARb/d is upregulated [19]. In mouse skin, PPARb/d is upregulated in direct response to the pro-inflammatory cytokine interferon-g (IFN-g) [16]. Recently, it has been demonstrated that PPARb/d mediates tumor necrosis factor (TNF)-induced survival of activated T-cells within psoriatic lesions [40]. This suggests that PPARb/d may actually contribute to the pathogenesis of psoriasis. However, in a mouse model of wound healing, TNFa via PPARb/d
promotes both epidermal differentiation and moderation of inflammation [16]. This mechanism involves ceramide as a second messenger in the TNF signaling cascade leading to upregulation of PPARb/d. So the role of PPARb/d as a mediator of inflammation and epidermal homeostasis appears to be highly dependent on the environment that the keratinocyte is in (e.g., wounded or inflamed), as well as on the species of animal model or the disease state. It is interesting to note that ceramide can play a role in the upregulation of PPARb/d [16]. It could be speculated that the increased synthesis and secretion of ceramides during epidermal differentiation could concomitantly increase the expression of PPARb/d. This may explain why PPARb/d is the most abundant PPAR found in normal skin [8, 19]. PPARa agonists such as clofibric acid and WY-14643 appear able to reduce inflammation by preventing inflammatory cell infiltrate. In mouse models of irritant and allergen contact dermatitis, these agonists reduced the amount of pro-inflammatory cytokines, TNFa and interleukin-1 (IL-1) [17]. These cytokines are key drivers of the inflammatory cascades that lead to the migration of inflammatory cells into affected tissues. In this model, the authors comment on the fact that the PPARa agonists were of similar potency as the corticosteroid clobetasol, an indication of the potential for PPAR therapies in treating inflammatory skin disease. The levels of inflammatory mediators – such as IL-1b, TNFa, IL-6, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) – increase with age, and aged animals have an increased sensitivity to inflammatory stimuli [35]. A key signaling pathway for both synthesis and activity of pro-inflammatory mediators is the nuclear factor-kB (NK-kB) pathway. Under normal conditions the NF-kB pathway is activated transiently, to enable an appropriate response to the stimulating challenge, such as injury or infection. As resolution of the insult proceeds, the pathway’s activity decreases. However, in aged animals there is evidence for constitutive activation of NF-kB, which may contribute to the acceleration of the aging process [41]. Activation of PPARa has been shown to repress NF-kB signaling and reduce production of IL-6 and IL-12 [42]. A recent article has proposed that the inhibitor of NF-kB kinase-alpha (IKKa) is a master regulator of epidermal differentiation [43]. The role of IKKa within the NF-kB signaling pathway is to phosphorylate inhibitors of NF-kB (IkBs), allowing translocation of NF-kB to the nucleus. Exactly how IKKa is involved in keratinocyte differentiation is not completely clear, but these data suggest that keratinocyte hyperproliferation may be driven, in part, by inflammatory pathways. This
Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin
appears to demonstrate that epidermal homeostasis is very closely linked to the inflammatory status of the skin, and that PPARs could be intricately involved in regulation of both. Exposure to UV light accelerates skin aging, and UV exposure induces skin inflammation, which is a major player in skin aging. While UVB will only penetrate to the epidermis, UVA can penetrate the skin to the dermis, where it will initiate a cascade of events that leads to the degradation of the dermal extracellular matrix. These changes take the form of increased collagen degradation, increased catabolic activity of dermal fibroblasts, and effects on the vasculature that promote the infiltration of inflammatory cells. This infiltrate adds to the catabolic environment within the dermis. Cytokines and chemokines are key to this process, as they orchestrate the response to the UV insult. As discussed above, many of the pro-inflammatory mediators act via the NF-kB intracellular signaling pathway. As such, PPARs are likely to be involved in mediating the inflammatory reaction to UV exposure. There is evidence that pre-treatment of skin with the PPARa agonist WY-14643 protects against UVB-induced erythema [44]. UVB can only penetrate as far as the epidermis; however, the ultimate effect of UVB irradiation in this model is an increase in erythema, which suggests keratinocyte-induced effects could be driving dermal vasculature changes that lead to the increased blood flow – in other words, an epidermal-induced dermal effect. Potential mediators for this epidermal driven process could be IL-8 and IL-6. IL-8 is a CXC chemokine known to be expressed by keratinocytes [45], and its function is to draw inflammatory cells out of the vasculature to the seat of an infection or injury. IL-6 is a prominent earlyphase cytokine, released in response to insult, which triggers many inflammatory cascades. Expression of IL-6 and IL-8 were both reduced by WY-14643 in UVB-irradiated keratinocyte cultures [44]. In addition to being induced in response to UV irradiation, IL-6 is known to be upregulated with age [46–48], raising the possibility that IL-6 may play a role in skin aging. IL-6 is a member of a family of cytokines known as the gp130 binding cytokines. They are grouped together as their receptor complexes share a common receptor subunit: gp130. The primary signaling pathway utilized by this family is the janus kinase/signal transducer and activation of transcription (JAK/STAT) pathway. It is known that members of the IL-6 family, which includes oncostatin M also present in skin [49, 50], can synergize with IL-1 or TNF to induce matrix-degrading enzymes [51]. Data from this and similar studies [52–54] suggest
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that low, physiologically relevant levels of the cytokines are enough to induce catabolic events that could contribute to the molecular causes of aged skin. The synergies between these cytokines occur at convergence points within their intracellular signaling pathways. There is evidence in the literature that activation of PPARs can repress the activity of the JAK/STAT pathway [55] and the AP-1 pathway [16]. This evidence, and the fact that PPARs may be able to regulate IL-6 expression in UV-induced inflammation and induce repressive pressures on NF-kB signaling, suggests that intervention or prophylactic treatment with PPAR therapies may be able to ameliorate some of the inflammation-driven causes of premature skin aging. Many of the natural ligands for PPARs are lipids; therefore, it is not surprising that some members of the bioactive lipid mediator families, prostaglandins, leukotrienes and thromboxanes, have been identified as PPAR ligands. One such mediator is leukotriene B4 (LTB4). The identification of LTB4 as a PPARa ligand came about during investigations to explore whether induction of PPARa activity would increase the catabolism of LTB4 [56]. The pro-inflammatory actions of LTB4 are controlled via its catabolism by peroxisomal enzymes; thus, increasing peroxisomes via PPARs was investigated. This was indeed the case; the authors demonstrated that PPARa knockout mice took longer to resolved LTB4induced inflammation. Prostagalandin E2 (PGE2) is a pro-inflammatory mediator that is also derived from the membrane-bound lipid arachidoninc acid. Cyclooxygenase enzymes (COX-1 and 2) are responsible for the initial cleavage of arachidonic acid, which, via intermediates, leads to the synthesis of PGE2 and other bioactive lipid mediators. PGE2 is required for UVB-induced inflammation and is the main prostaglandin produced by keratinocytes [57]. While there is no evidence to date that PGE2 can bind directly to PPARs, there is evidence in the literature that activation of PPARg can suppress the transcriptional activation of COX-2 and subsequent synthesis of PGE2 [58]. The PPARg ligands tested in this study were ciglitazone, troglitazone, and 15-deoxy-D12,14 prostaglandin J2 (15dPGJ2). 15d-PGJ2 is another member of the prostaglandin family; however, binding of 15d-PGJ2 to its target receptors tends to elicit anti-inflammatory events. 15d-PGJ2 is a natural ligand for PPARg, and its effects on the synthesis of prostaglandins by suppressing the synthesis of COX2 are PPARg-dependent. 15d-PGJ2 can also mediate antiinflammatory activity independent of PPARg [55]. As discussed above, PPARs can act upon the JAK/STAT pathway to reduce inflammation. It appears that 15d-PGJ2 can reduce the activity of this pathway [59, 60].
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CLA: A Naturally Occurring Lipid with PPAR Activity Dietary literature suggests that Conjugated Linoleic Acid (CLA) provides a number of physiologic health benefits to the immune system from supplements, which may involve regulation of prostaglandin, cytokine, and PPAR pathways. (For reviews, see [60–62] and references therein.) CLA may be extracted from sunflower or safflower oil, and is naturally present in a number of foods, including dairy products (milk, cheese, butter, yoghurt) and meats (beef, lamb). CLA is a collective term for positional and geometric (cis/trans) isomers of linoleic acid (C18:2), in which two double bonds are conjugated (e.g., separated by one carbon-carbon single bond). Principal isomers produced from the conjugation of linoleic acid are cis-9, trans-11 CLA and trans-10, cis-12 CLA (> Fig. 21.1). CLA has been shown to be a natural ligand for all three of the PPAR receptors [61, 63, 64]. While most studies on CLA have been directed at understanding the dietary benefits of CLA supplementation, the potential to use CLA as topically applied cosmetic ingredient to improve the appearance of photoaged skin is being investigated. Previous research has demonstrated that CLA delivers a variety of anti-aging benefits to skin, including a
reduction in the appearance of overall photodamage, mottled hyperpigmentation, lines and wrinkles, and coarse wrinkles [29].
Clinical Studies with CLA: Evidence of Anti-aging Benefit from Topical Application Photodamaged Split-Application Facial Studies Healthy female subjects (ages 40–70 years), with Fitzpatrick skin type I–III and a moderate degree of expert-assessed photodamage, were enrolled in Institutional Review Boardapproved, randomized, split-face application studies. A total of 64 subjects were enrolled to complete two facial photodamage studies. Subjects provided informed consent to participate in each study. Subjects were excluded if they had previous reactions to a-hydroxyl acids, retinoids, fragrances, or soaps. Test products were a cream containing 3% CLA and its vehicle. Subjects applied each test product (approximately 0.35 g for each side at each application) to each side of their face twice daily for 12–16 weeks. Subjects were instructed to use their current cleansing regimen throughout the study, and agreed to discontinue use of
. Figure 21.1 Conjugated Linoleic Acid (CLA). CLA is a collective term for positional and geometric (cis/trans) isomers of linoleic acid (C18:2), in which two double bonds are conjugated (e.g., separated by one carbon-carbon single bond). Principal isomers produced from the conjugation of linoleic acid are cis-9, trans-11 CLA and trans-10, cis-12 CLA
Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin
their normal moisturizers and other anti-aging products (e.g., masks, eye creams, or gels) throughout the study. Expert visual assessment of photodamage attributes in the periorbital region (rated on a 0–9 scale, where 0 = none, 1–3 = mild, 4–6 = moderate, and 7–9 = severe) was performed at baseline and at monthly time points by an expert clinical evaluator. Fine lines in the crow’s feet area (periorbital), fine lines under the eye, and overall photodamage were assessed on each side of the face. Analysis of between-treatment differences observed by expert visual assessment was calculated using the Wilcoxon signed-rank test. Digital photos were obtained at baseline and Weeks 12 and 16 for each side of the face (approximately 45 views) to record improvement in the appearance of photodamage using the VISIA-CR1 photography system (Canfield Scientific, Inc., Fairfield, NJ, USA). Color calibration cards were used to ensure and monitor quality of the images throughout the studies. 25 subjects completed the first facial study, which was a 16-week double-blind product application study. The CLA cream significantly improved the appearance of photodamage attributes from baseline conditions (> Figs. 21.2–21.4) as assessed by an expert clinical evaluator. Fine lines in the crow’s feet and under-eye areas significantly improved from baseline conditions at Weeks 12 and 16 (p < 0.05). By Week 12, the CLA cream provided significant improvement in expert visual assessment of photodamage attributes compared with its vehicle cream (> Figs. 21.2–21.4), which continued at Week
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16. Sample photos of individual subjects (baseline compared with Week 16) are shown in > Figs. 21.5 and > 21.6. Clinical improvement of fine lines and wrinkles in the periorbital region were observed, as well as improvement in overall appearance. 39 subjects completed the second facial study, which was a 12-week double-blind product application study. Expert visual evaluation of photodamage was performed at baseline and after 12 weeks of product application. Objective measurements with 3D facial scans were obtained using the Cyberware™ 3030/HIREZ facial scanner (Cyberware, Inc., Monterey, CA, USA) at baseline and at Weeks 2, 4, 6, 8 and 12. Virtual 3D images were obtained via facial scanning, across a 15 cm vertical line, 90 section, sampled at a rate of 0.2 . The resulting scans captured images approximately from ear to ear on each subject’s face. The vertical scan data captured images approximately from subjects’ chins upward to mid-forehead, depending on the size of the individual’s face. The nasolabial fold lines, characterized by length and depth parameters were quantified using Echo-TK/Cyscan™ software. At Week 12, fine lines in the crow’s feet area, under the eye, and overall photodamage significantly improved from baseline condition with the CLA cream (> Fig. 21.7). In addition, the CLA-treated side was significantly more improved than its vehicle-treated control side, and the vehicle-treated side was not significantly improved for any visual assessment at Week 12. A subset of the panel (N = 12) were measured using objective 3D facial
. Figure 21.2 Clinical evaluation of periorbital fine lines. Compared with its vehicle, CLA cream significantly improved the appearance of periorbital fine lines (P < 0.05)
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. Figure 21.3 Clinical evaluation of fine lines under the eye. Compared with its vehicle, CLA cream significantly improved the appearance of fine lines under the eye and on the upper cheek (P < 0.05)
. Figure 21.4 Clinical evaluation of overall appearance. Compared with its vehicle, CLA cream significantly improved overall appearance (P < 0.05)
scanning measurements, via measurement of nasolabial fold lines (length in mm). The CLA-treated side showed significant improvement from baseline condition at Weeks 8 and 12; the vehicle-treated side showed no significant improvement at any week (> Fig. 21.8).
Biopsy Study on Photodamaged Forearms Healthy female subjects (ages 40–65 years) with Fitzpatrick Skin Type I–III and mild to moderately photodamaged volar forearms, provided informed consent to
Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin
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. Figure 21.5 Courtesy at (a) baseline and (b) Week 16 for individual subject. CLA cream significantly improved the appearance of photodamage attributes such as fine lines, wrinkles, and overall appearance. Application of the CLA cream has reduced the length and depth of this subject’s crow’s feet. The fine lines in this subject’s under-eye area have also decreased in depth and become less apparent
. Figure 21.6 Courtesy at (a) baseline and (b) Week 16 for individual subject. CLA cream significantly improved the appearance of photodamage attributes such as fine lines, wrinkles, and overall appearance. The depth of this subject’s fine lines in the crow’s feet area have greatly decreased with use of the CLA cream. An improvement in photodamage is also seen in the subject’s overall appearance, particularly in the lower cheek area
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Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin
. Figure 21.7 Clinical evaluation of CLA cream compared to its vehicle at Week 12, by expert assessment of photodamage. Significant improvement was observed with CLA for all attributes, compared to baseline skin condition. Significant improvement favoring CLA over its vehicle was observed for all attributes
. Figure 21.8 Objective 3D assessment of CLA compared to its vehicle, using Cyberware Facial Scanning
Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin
participate in a 21-day repeat (9 24 h) patch test, followed by a dermatologist-collected punch biopsies test. 12 total subjects were enrolled, and completed all phases of the study. The test methodology and evaluation results with CLA were previously described by Shingleton et al. [65]. Test products included CLA formulated into a simple vehicle, the vehicle, and 0.05% retinoic acid as a positive control. Test products were applied to the skin under occlusive patch for 24 h, with 24-h recovery gaps over the course of 21 days. On the last day of the study the consulting dermatologist took full thickness punch biopsies from each site. Biopsies were formalin-fixed, processed for paraffin wax embedding, and sectioned. Sections were stained for H&E and orcein. Further sections were subjected to immunohistochemical analysis for filaggrin expression. The degree of filaggrin staining was assessed by a trained expert evaluator. The CLA and retinoic acid responses were normalized to degree of staining with the vehicle control, and reported as mean increased percentage of responses. Erythema was assessed by the consulting dermatologist on a 0–4 scale (where 0 = none and 4 = severe). The analysis of biopsies from subjects treated with CLA showed increased keratinocyte proliferation and epidermal thickness (9 out of 12). A significant increase in the expression of filaggrin following CLA cream application was also observed, suggesting a concomitant increase in epidermal differentiation. Detailed histological evaluation of the dermis showed evidence of stimulation of extracellular matrix turnover. In addition, increased removal of elastotic material and a modest increase in damaged collagen degradation was observed in all subjects treated with the CLA cream. After removal of the final patch, there was no significant difference in edema observed between the vehicle and the CLA cream. However, as expected, the retinoic acid control showed significantly increased erythema/edema grades compared to the measured response of the vehicle.
Conclusion Clinically, CLA – a cosmetic PPAR lipid – has recently been shown to improve the appearance of photodamage [29], such as reduced fine lines and wrinkles, overall appearance, and the appearance of coarse rhytides in the nasolabial fold area, in vehicle-controlled photodamaged facial studies as described above. CLA cream increased epidermal proliferation, leading to an increase in the
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thickness of the epidermis in a photodamaged forearm test, with no significant edema, whereas application of retinoic acid (0.05%) induced significant irritation/ edema. The increase in keratinocyte proliferation was accompanied by an increase in differentiation, as judged by the significant increase in filaggrin expression and a thickening of the stratum granulosum, which is likely to result in improved epidermal quality. The magnitude of the effects described above is significant and similar to the effects described when PPARs are activated in models of epidermal development and disease. There is an apparent increase in epidermal proliferation [10, 11], and a concomitant increase in differentiation [12, 13]. Evidence of dermal effects following CLA treatment in the photodamaged forearm model is well demonstrated; dermal fibroblasts have been shown to respond to activators of PPARa in vitro [66]; however, there is no evidence to suggest that topically applied PPAR agonists can activate fibroblasts directly. It is possible that by inducing beneficial effects on epidermal homeostasis, CLA also triggers an epidermal-induced dermal response that may lead to the observed dermal changes. The role of the PPARs in skin homeostasis is now widely accepted, as is their benefit in treating hyper-proliferative and inflammatory skin disorders. Keratinocytes can respond to the environment they are in via signaling pathways that utilize PPARs; this includes both cell cycle control and inflammatory pathways. Akin to their role in systemic lipid control, PPARs in skin could be regarded as the keratinocyte sensors leading to homeostatic control of inflammation as well as differentiation. As such, they have high potential as targets for cosmetic and therapeutic intervention strategies. The significant improvement in photodamaged attributes with no irritation have been summarized here for CLA, a natural ligand for PPAR, in vehicle controlled, double-blind clinical trials.
Acknowledgment The authors would like to thank Susan Krein, Vickie Foy, Priya Vaidyanathan, and Robert Marriott for their contributions to this research.
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Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin 36. Boyd AS. Thiazolidinediones in dermatology. Int J Dermatol. 2007;46:557. 37. Dahten A, Koch C, Ernst D, Schnoller C, Hartmann S, Worm M. Systemic PPAR gamma ligation inhibits allergic immune response in the skin. J Invest Dermatol. 2008;128:2211. 38. Michalik L, Wahli W. Peroxisome proliferator-activated receptors (PPARs) in skin health, repair and disease. Biochim Biophys Acta-Mol Cell Biol Lipids. 2007;1771:991. 39. Demerjian M, Man MQ, Choi EH, Brown BE, Crumrine D, Chang S, Mauro T, Elias PM, Feingold KR. Topical treatment with thiazolidinediones, activators of peroxisome proliferator-activated receptorgamma, normalizes epidermal homeostasis in a murine hyperproliferative disease model. Exp Dermatol. 2006;15:154. 40. Al YN, Romanowska M, Krauss S, Schweiger S, Foerster J. PPAR delta is a type 1 IFN target gene and inhibits apoptosis in T cells. J Invest Dermatol. 2008;128:1940. 41. Spencer NF, Poynter ME, Im SY, Daynes RA. Constitutive activation of NF-kappa B in an animal model of aging. Int Immunol. 1997;9:1581–1588. 42. Poynter ME, Daynes RA. Peroxisome proliferator-activated receptor alpha a´Activation modulates cellular redox status, represses nuclear factor-kappa B signaling, and reduces inflammatory cytokine production in aging. J Biol Chem. 1998;273:32833–32841. 43. Liu B, Zhu F, Xia X, Park E, Hu Y. A tale of terminal differentiation: IKKalpha, the master keratinocyte regulator. [In Process Citation]. Cell Cycle (Georgetown, Tex 8). 2009;8(4):527–531. 44. Kippenberger S, Loitsch SM, Grundmann-Kollmann M, Simon S, Dang TA, Hardt-Weinelt K, Kaufmann R, Bernd A. Activators of peroxisome proliferator-activated receptors protect human skin from ultraviolet-B-light-induced inflammation. J Invest Dermatol. 2001;117:1430–1436. 45. Coquette A, Berna N, Vandenbosch A, Rosdy M, De Wever B, Poumay Y. Analysis of interleukin-1 alpha (IL-1 alpha) and interleukin-8 (IL-8) expression and release in vitro reconstructed human epidermis for the prediction of in vivo skin irritation and/or sensitization. Toxicology In Vitro. 2003;17:311–321. 46. Daynes RA, Araneo BA, Ershler WB, Maloney C, Li GZ, Ryu SY. Altered regulation of interleukin-6 production with normal aging. J Immunol. 1993;150:A285. 47. Ershler WB. Interleukin-6 - A cytokine for gerontologists. J Am Geriatr Soc. 1993;41:176–181. 48. Ershler WB, Keller ET. Age-associated increased interleukin-6 gene expression, late-life diseases, and frailty. Ann Rev Med. 2000; 51:245–270. 49. Boniface K, Diveu C, Morel F, Pedretti N, Froger J, Ravon E, Garcia M, Venereau E, Preisser L, Guignouard E, Guillet G, Dagregorio G, Pene J, Moles JP, Yssel H, Chevalier S, Bernard FX, Gascan H, Lecron JC. Oncostatin M secreted by skin infiltrating T lymphocytes is a potent keratinocyte activator involved in skin inflammation. J Immunol. 2007;178:4615. 50. Yu M, Kissling S, Freyschmidt PP, Hoffmann R, Shapiro J, McElwee KJ. Interleukin-6 cytokine family member oncostatin M is a hairfollicle-expressed factor with hair growth inhibitory properties. Exp Dermatol. 2008;17:12. 51. Rowan AD, Koshy PJT, Shingleton WD, Degnan BA, Heath JK, Vernallis AB, Spaull JR, Life PF, Hudson K, Cawston TE. Synergistic effects of glycoprotein 130 binding cytokines in combination with interleukin-1 on cartilage collagen breakdown. Arthritis Rheum. 2001;44:1620–1632. 52. Cawston TE, Curry VA, Summers CA, Clark IM, Riley GP, Life PF, Spaull JR, Goldring MB, Koshy PJT, Rowan AD, Shingleton WD. The
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6 Physiological Variations During Aging Ge´rald E. Pie´rard . Philippe Paquet . Emmanuelle Xhauflaire-Uhoda . Pascale Quatresooz
Introduction In affluent societies, an extraordinary shift has taken place over the past decades in the age distribution of the populations. Indeed, older people progressively represent a growing segment of the demographic profile. Nobody can escape from aging. Such evolution is correlated with the individual loss of youthful appearance, which in turn has important social implications. As a result, any newer medical and cosmetic anti-aging modalities are avidly watched by the aging individuals. Moreover, middle-aged and younger subjects may show a craze for cosmetic dermatology beginning at the onset of early signs of wear and tear. Hopefully, breakthroughs in cosmetic procedures and novel treatments fulfill some of the expectations and promises. In addition, technological advances at the forefront of the management of skin aging are supported by a better understanding of the relationships among skin biology, skin physiology, and the ultimate clinical appearance. Both physical growth and senescence are characterized by cumulative progression of interlocking biologic events. They may proceed at some time in life as if they were evolving in tandem. Globally, aging is a physiological process corresponding to a progressive impairment in the homeostatic and adaptive homeodynamic capacities of the body systems, ultimately increasing the susceptibility and vulnerability to environmental threats and internal changes. While aging is associated with a slower rate of cutaneous wound healing, it may be paradoxically associated with an improvement in the quality of scarring. The global aging process is quite complex. It becomes severe in cases where the impaired skin has lost most of its protective biological and mechanical functions. The atrophy becomes considerably disabling when the aspect of the ‘‘transparent skin’’ or ‘‘dermatoporosis’’ is reached [1]. The corresponding clinical manifestations encompass a series of markers of fragility including senile purpura, stellate pseudoscars, and skin thinning. Skin fragility leads to frequent lacerations following minor traumas. A varied combination of delayed wound healing, nonhealing atrophic ulcers, subcutaneous bleeding, and dissecting hematomas is at the origin of large necrotic areas.
From Global to Molecular Aging and Back Again All living organisms are engaged in multifaceted aging processes that show interspecies variations. Two complementary classifications of life histories are of major importance in the biological definition of aging. The first classification distinguishes species that show a clear distinction between germ cells and somatic tissues from those that do not. The second classification distinguishes the semelparous species reproducing only once in their lifetime, from the iteroparous species, which reproduce repeatedly. It is a mistake to regard the postreproductive end of life of semelparous species, which usually occurs in a highly determinate fashion, as being comparable with the more protracted process of senescence in iteroparous species. There is evidence that aging progresses differ among individuals of the same age. In addition, each and every organ of the human body develops and fails at its own rate, which is referred to as its age [2]. Senescence is heterogeneous at other levels including tissues, cells, and subcellular structures [3]. Intracellular and extracellular molecular compounds are involved differently in aging. However, within each organ system, aging manifests as a progressive, almost linear reduction in maximal function and reserve capacity. Although some aspects of aging appear as a predetermined programmed process, many of the age-associated physiological decrements result in part from environmental insults and/or from endogenous failures. The global but heterogeneous aging occurs throughout the entire body from the time of about 30–45 years of age (> Table 6.1). To make the situation more complex, there is regional variability in skin aging over the body. It is indeed quite evident that at any time in adult life the face, scalp, forearms, trunk, and other body sites show different manifestations or stages of aging. In addition, scrutinizing skin aging at the tissue level (epidermis, dermis, hypodermis, hair follicle), and further at the cellular level (keratinocyte, melanocyte, fibroblast, dermal dendrocyte, endothelial cells, etc.) shows a patchwork of aging severity.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_6, # Springer-Verlag Berlin Heidelberg 2010
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Physiological Variations During Aging
. Table 6.1 Core age markers of each of the body system (Adapted from Braverman [2]) Aging type
Decline in
Average onset age
Electropause
Electrical activity of brain waves
45
Biopause
Neurotransmitters
Dopamine 30, Acetylcholine 40, GABA 50, Serotonin 60
Pineal pause
Melatonin
20
Pituitary pause
Hormone feedback loops
30
Sensory pause
Touch, hearing, vision, taste, and smell sensitivity
40
Psychopause
Personality health and mood
30
Thyropause
Calcitonin and thyroid hormone levels
50
Parathyropause
Parathyroid hormone
50
Thymopause
Glandular size and immune system
40
Cardiopause/vasculopause
Ejection fraction and blood flow
50
Pulmonopause
Lung elasticity and function with increase in blood pressure
50
Adrenopause
DHEA
55
Nephropause
Erythropoietin level and creatinine clearance
40
Somatopause
Growth hormone
30
Gastropause
Nutrient absorption
40
Pancropause
Blood sugar level
40
Insulopause
Glucose tolerance
40
Andropause
Testosterone in men
45
Menopause
Estrogen, progesterone, and testosterone in women
40
Osteopause
Bone density
30
Dermopause
Collagen, vitamin D synthesis
35
Onchopause
Nail growth
40
Uropause
Bladder control
45
Genopause
DNA
40
Cellular Senescence in Perspective Several basic cellular mechanisms are involved in the aging process: the telomere shortening, the oxidative stress, the mitochondrial dysfunction, and a series of genetic mechanisms. Granted that death is the ultimate failure of the organisms, the aging process itself is considered to bring about the termination of the replicative ability of cells as the individual becomes progressively older. The age of any tissue appears to be reflected in the behavior of their cultured skin-derived cells [4]. Replicative senescence of human cells is thus related to and perhaps caused by the exhaustion of their proliferative potential. At each
cell division, cells lose some of their telomere repeats corresponding to specific DNA sequences at the end of linear DNA [5]. According to the telomere hypothesis, somatic cells lack a sustained activity of telomerase to maintain the telomere repeats allowing replication. The telomere shortening is attributed to the accumulation of DNA single-strand breaks induced by oxidative stress. Since telomere length predicts the replicative capacity of cells, it is thought to provide the best biomarker for cellular aging [6]. Aging is associated to free radical damage by a variety of reactive oxygen species (ROS) including superoxide and hydroxyl radicals, as well as other activated forms of oxygen such as hydrogen peroxide and singlet oxygen [7].
Physiological Variations During Aging
In the intracellular machinery, mitochondria are the primary sites of production of ROS. Enzymes that minimize oxidative injury include superoxide dismutase, catalase, gluthatione peroxidase, glutathione transferases, peroxidases, and thiol-specific antioxidant enzymes. In addition, ROS play a role in normal signaling processes. Their generation is essential to maintain homeostasis and cellular responsiveness [8]. The so-called stress-induced premature senescence (SIPS) occurs following a series of sublethal stresses including those induced by H2O2, other ROS, and a variety of other chemicals [9]. Of note, cells engaged in replicative senescence share common features with cells involved in SIPS [9]. Thus, SIPS could be a mechanism of the in vivo accumulation of senescent-like cells in the skin [10]. Mitochondria are both producers and targets of oxidative stress, thus forming the basis for the mitochondria theory of aging. With advanced age, the activity of the mitochondrial respiratory system declines. The integrity of the mitochondrial DNA is reduced, eventually leading to apoptosis [11–13]. It is acknowledged that the alterations in the mechanisms of apoptosis result from genetically programmed features and oxidative stress as well [14, 15]. Cellular senescence and cancer are closely related by several biological aspects including p53 mutation [16, 17], telomere shortening [5, 18], vitamin A depletion [19], and defects in intercellular communications [20].
6
The age-related mottled faint (subclinical) melanoderma might be a predictive sign for a skin carcinoma-prone condition [21–23].
Clinically Relevant Skin Aging Mechanisms Human aging may be perceived as one single chronologic process of physiological decline with age. However, this process exhibits multiple facets altering differently the organs, tissues, and cells. This is particularly obvious in the skin. For years, the understanding of aging skin has benefited from the distinction between the intrinsic chronologic aging and photoaging [24]. According to this concept, the changes observed in the aging skin appearance reflect two main processes (> Table 6.2). First, the intrinsic changes in the aging skin are caused by the passage of time modulated by hereditary factors, along with modifications occurring inherently in the structure, physiology, and mechanobiology. Second, photodamage is a result of the cumulative exposure of the skin to ultraviolet (UV) light and near-infrared radiations (IRA) [25]. Clinically, these two types of aging may manifest differently, with intrinsic aging being responsible for dry, pale, and smooth-to-finely wrinkled skin. By contrast, photoaging gives rise to coarse, roughened, and deeply wrinkled skin accompanied by pigmentary changes such
. Table 6.2 Comparison of intrinsic aging and photoaging Feature
Intrinsic aging
Photoaging
Clinical appearance
Smooth texture, unblemished surface. Fine Nodular, leathery surface. Sallow complexion wrinkles. Some deepening of skin surface markings yellowish mottled, pigmentation. Coarse wrinkles Some loss of elasticity, redundant skin Severe loss of elasticity
Epidermis
Thin and viable
Marked acanthosis, cellular atypia, skin field carcinogenesis, most skin cancers
Elastic tissue
Increased, but almost normal
Tremendous increase, altered as elastotic amorphous mass
Collagen
Thick, disoriented bundles
Marked decrease of bundles and fibers
Glycosaminoglycans Slightly decreased
Markedly increased
Reticular dermis
Thinner fibroblasts decreased, inactive mast cells decreased, no inflammation
Thickened, elastosis. Fibroblasts increased, hyperactive Mast cells markedly increased, mixed inflammatory infiltrate
Papillary dermis
No Grenz zone
Solar elastosis with Grenz zone
Microvasculature
Moderate loss
Great loss, abnormal, and telangiectatic
Subcutaneous fat
Focal shrinkage. Focal hypertrophy
Unaffected
Hair
Hair thinning Graying hair. Hypertrichosis
Discoloration
47
48
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Physiological Variations During Aging
as solar lentigines and mottled melanosis. Differences between these two types of aging can be seen within one individual when comparing an area of skin commonly exposed to the sun, for example, the face, the neck, and the dorsal forearms, with an area commonly masked from the sun, for example, buttock skin. This concept based on a duality in skin aging has been challenged because it may appear as an oversimplification in clinical practice [26]. In addition, recent evidence indicates that chronologically aged and UV-irradiated skin share important molecular features [7, 8, 27, 28]. Oxidative stress is thought to play a central role in initiating and driving the signaling events that lead to cellular response following UV and IRA exposures. These features suggest that nonionizing radiations accelerate many key aspects of the chronological aging process in human skin [25]. In order to better cope with the diversity of clinical presentations of skin in the elderly, another classification of skin aging in seven distinct types was offered (> Table 6.3). The most important categories included the endocrine and overall metabolic status, the past and present lifestyle, and several environmental threats, including cumulative UV and IRA exposures, and repeated mechanical solicitations by muscles and external forces including earth gravity [26]. In this framework, the past history of the subject is emphasized. Accordingly, the global aging is considered to represent the cumulative or synergistic effects of specific features, each of them being independent from the others. Such a concept allows to individualize or integrate typical processes including among others climacteric aging and smoking effects. Increased awareness of the distinct age-associated physiologic changes in the skin may allow for more effective and specific skin care regimens, preventive measures, and dermatologic treatment strategies in the elderly. As a consequence, the immutability of diverse factors in skin aging was challenged [3, 29]. However, factors of skin
aging share some common mechanisms [30]. For instance, molecular mechanisms imply hyaluronate-CD44 pathways in the control and maintenance of epithelial growth and the viscoelastic properties of the extracellular matrix offer new opportunities for preventive intervention [1].
Environmental Aging and Photoaging Environmental influences produce obvious alterations in the texture and quality of the skin, the major extrinsic insults being chronic exposure to UV and IRA [25]. The action spectrum of photodamages is not fully characterized. The cumulative effects from repeated exposures to suberythemal doses of UVB, UVA, and IRA in human skin are involved in these processes [25, 31]. The role of UVB in elastin promoter activation in photoaging is obvious. In addition, UVA significantly contributes to long-term actinic damage, and the spectral dependence for cumulative damages does not parallel the erythemal spectrum for acute UV injury in human beings. The earliest detectable response of the skin cell to UV irradiation is the activation of multiple cytokine and growth factor cell surface receptors including epidermal growth factor receptor (EGF-R), TNF-a receptor, platelet-activating factor (PAF receptor) insulin receptor, interleukin (IL)-1 receptor, and platelet-derived growth factor (PDGF) receptor [27]. Nonionizing radiations initiate a number of cellular responses, including ROS production within both dermal and epidermal cells. The so-called stress-induced parameter senescence (SIPS) phenomenon is engaged [9]. More specifically, cultures of keratinocytes derived from donors of different ages and from paired sun-exposed and sunprotected sites of older donors demonstrate that both chronological aging and photoaging affect quite distinctly
. Table 6.3 Cutaneous aging types (Adapted from Pie´rard [26]) Aging type
Determinant factor
Genetic
Genetic (premature aging syndromes, phototype-related, ethnic background)
Chronologic
Time
Actinic
Ultraviolet and infrared irradiations
Behavioral
Tobacco, alcoholic abuse, drug addiction, facial expressions
Endocrine
Pregnancy, physiological and hormonal influences (ovaries, testes, thyroid)
Catabolic
Chronic intercurrent debilitating disease (infections, cancers), nutritional deficiencies
Gravitational
Earth gravity
Physiological Variations During Aging
some gene expressions. For instance, chronological aging strikingly increases the baseline expressions of the differentiation-associated gene SPR2 (small proline-rich protein) and of the interleukin (IL)-1 receptor antagonist gene. By contrast, it has relatively little effect on the UV-inducibility of several other genes including the protooncogenes, c-myc and c-fos, the GADD 153, a gene inducible by growth arrest and DNA damage, and the IL-1a and 1L-b genes. Photoaging appears different because it increases the UV-inducibility of c-fos while decreasing the baseline expression of the differentiationassociated genes IL-1ra and SPR2 [32, 33]. The physiological consequences of photodamages occur at variable pace on the different skin structures. For instance, skin loosening and solar elastosis exhibit clinical manifestations independently from the severity in the mottled faint melanoderma [21]. IR are nonionizing, electromagnetic radiations accounting for more than half of the solar energy that reaches the human skin. The IR wavelengths ranges between 760 nm and 1 mm, and are further divided into IRA, IRB, and IRC. While IRB and IRC do not penetrate deeply into the skin, more than 65% of IRA reaches the dermis. Human skin is increasingly exposed to IRA radiation. The most relevant sources are: (a) natural solar radiation consisting of over 30% IRA, (b) artificial IRA sources used for therapeutic or wellness purposes, and (c) artificial UVA sources supplemented with IRA. As part of natural sunlight, IRA significantly contributes to extrinsic photoaging. Photoaging affects both the epidermis and dermis. The epidermis becomes more atrophic than sun-protected areas often with disordered keratinocyte maturation. This may represent an early step in actinic field carcinogenesis. Key histological features of photoaged skin are most apparent in the dermis where the extracellular matrix is altered in its composition [34]. The collagen network is responsible for most skin strength and resiliency, and is intimately involved in the expression of photoaging. The major fibrillar collagen components of the dermis belong to the type I and III collagens. In photoaged human skin, precursors of both proteins are markedly reduced in the papillary dermis, and their reduction correlates with the clinical severity of photoaging [35]. This reduction results from a combination of decreased procollagen biosynthesis contrasting with an increased enzymatic breakdown by matrix metalloproteinases (MMPs) [36]. Furthermore, collagen breakdown products negatively influence procollagen biosynthesis by fibroblasts [37]. Fibrillar collagens are closely associated with the small chondroitin sulfate proteoglycan, and decorin. Its
6
distribution closely mirrors that of type I collagen in the dermis, regardless of level of extrinsic aging [38]. Decorin makes connections between the fibrillar collagens and the microfibril-forming type VI collagen with further interaction with type IV collagen in the basement membrane at the dermo-epidermal junction. Therefore, type VI collagen is likely to play an important physiological role in the organization of the dermal matrix. It is abundant in the papillary dermis and seems little affected by photoaging [39]. Type VII collagen is the major constituent of anchoring fibrils below the basement membrane providing cohesiveness between the epidermis and the dermis. In photoaged skin, the number of anchoring fibrils along the basement membrane is reduced, thus increasing the potential for fragility and blistering in photoaged skin [40]. It was reported to be involved in the mechanism of wrinkle formation. The elastic fiber network is responsible for recoil and elasticity of the skin. The process of elastic fiber formation is under tight developmental control, involving tropoelastin deposition on a preformed framework made of fibrillin-rich microfibrils. Mature elastic fibers are encased in fibrillin and form a continuous network throughout the dermis. The elastic fiber network comprises thick elastin-rich fibers within the reticular dermis, finer fibers with reduced elastin content in the lower papillary dermis, and a meshwork of discrete fibrillin-rich microfibrillar bundles, with only discrete elastin, in the upper papillary dermis merging with the dermo-epidermal junction. Fibrillin is a product of both fibroblasts and keratinocytes. The elastic fiber network is considerably disrupted and clumped in chronically photoaged skin. First, photoaged skin contains abundant amounts of dystrophic elastotic material in the reticular dermis [41], which is immunopositive for tropoelastin, fibrillin, lysozyme, and immunoglobulins [42] Versican, a large chondroitin sulfate proteoglycan, appears to be regulated along with dystrophic elastin resulting in a relative increase in photoaging. The fibrillin-rich microfibrils are markedly altered since the early stages of photoaging [43]. All structural changes found in the dermis particularly affect the biomechanical properties of the skin. A cutaneous extrinsic aging score was derived from the difference between comparative photoexposed and photoprotected areas [44].
Phototype and Ethnicity-Related Aging The most obvious ethnic skin difference relates to skin color, which is dominated by the presence of melanin [45, 46]
49
50
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Physiological Variations During Aging
affording relative photoprotection. The rate of skin aging varies between different racial groups, however, although none of the groups escape the photoaging process. Generally, Caucasians present an earlier onset and a deeper skin wrinkling and sagging than other skin phototypes. In general, increased pigmentary problems develop in skin of color, although East Asians living in Europe and North America exhibit less pigment spots with age. Induction of a melanotic response to sun exposure is thought to occur through signaling by the protease-activated receptor (PAR)-2, which together with its activating protease is increased in the epidermis of subjects with skin of color [47]. Changes in skin biophysical properties with age demonstrate that subjects of dark complexion keep younger skin properties compared with the more lightly pigmented individuals.
. Table 6.4 Neuroendocrine receptors active in the skin (Adapted from Quatresooz [50]) 1. Adrenergic receptors 2. Androgen and estrogen receptors 3. Calcitonin gene-related peptide receptor (CGRP-R) 4. Cholinergic receptors 5. Corticotropin-releasing hormone and urocortin receptors (CRH-R) 6. Glucocorticoid and mineralocorticoid receptors 7. Glutamate receptors 8. Growth hormone receptor (GH-R) 9. Histamine receptors 10. Melanocortin receptors (MC-R) 11. Miscellaneous neuropeptide receptors 12. Miscellaneous receptors
Endocrine Aging
13. Neurokinin receptors (NK-R) 14. Neutrophin receptors (NT-R)
Skin is recognized as a hormone-dependent organ [48–51]. Irrespective of age, most of the skin components are under the physiological control of endocrine and neuroendocrine factors (> Table 6.4). The whole endocrine system is affected by the global aging process. Like any other system in the body, aging of the hormonal functions basically results in deteriorations expressed by deficiencies, which in turn influence the aging machinery operative in the skin [50]. Quite distinct are the skin manifestations of some endocrinopathies, which mimic or interfere with skin aging [48–51]. They are mostly related to the declined activity of the pituitary gland, adrenal glands, ovaries, and testes. Some hormones and neurotransmitters are synthesized by nerves, as well as by epithelial and dermal cells in the skin (> Table 6.5). A number of environmental and intrinsic factors regulate the level of the cutaneous neuroendocrine system activity. Solar radiation, temperature, environmental moisture, and diverse chemical and biological xenobiotics represent important environmental factors. Some internal mechanisms affecting the neuroendocrine system of the skin may be generated in reaction to some environmental signals or result from local biological rhythms, or from local or general disease processes [49]. The paradigm of deleterious hormonal effects is represented by the induction of skin atrophy by corticosteroids. Cushing syndrome and iatrogenic effects of topical and systemic corticotherapy are equally involved. Corticosteroids are known to regulate the expression of genes
15. Opioid receptors 16. Parathormone (PTH) and PTH-related protein (PTHrP) receptors 17. PRL and LH-CG receptors (LH/CG-R) 18. Serotonin receptors 19. Thyroid hormone receptors 20. Vasoactive intestinal peptide receptor (VIP-R) 21. Vitamin D receptor (VDR)
. Table 6.5 Hormones and neurotransmitters produced by the skin (Adapted from Quatresooz [50]) 1. Hypothalamic and pituitary hormones 2. Neuropeptides and neurotrophins 3. Neurotransmitters/neurohormones 4. Other steroid hormones 5. Parathormone-related protein 6. Sex steroid hormones 7. Thyroid hormones
encoding collagens I, III, IV, V, decorin, elastin, MMPs 1, 2, 3, tenascin, and tissue inhibitors of MMPs 1 and 2 [52]. However, the precise molecular mechanisms of skin atrophy induced by corticosteroids are not yet known. The corticosteroid-induced atrophy can be one
Physiological Variations During Aging
of the most severe form of skin aging corresponding to dermatoporosis [1]. The most important endocrine compound produced by the skin is vitamin D, which is a regulator of the calcium metabolism and exhibits other systemic effects as well. For example, epidemiological evidence suggests that sunlight deprivation with associated reduction in the circulating level of vitamin D3 may result in increased incidence of carcinomas of the breast, colon, and prostate [53]. Vitamin D3 and its analogs also modulate the biology of keratinocytes and melanocytes of the skin in vivo [54]. Growth hormone (GH) is secreted by the pituitary gland under the control of several hypothalamic and peripheral modulators that exert either positive or negative influences [40]. The final balance among the modulating factors determines the pulsatile and circadian secretion of GH. Moreover, physiological changes occurring during aging affect the GH secretion. The peripheral effects of GH are mainly exerted by insulin-like growth factor (IGF), produced by the liver upon GH stimulation. The circulating IGF-1 is bioavailable and functionally active depending upon its binding with the IGF-binding proteins (IGF-Bps). Skin is a target of the GH-IGF system that exerts a significant influence on the dermal and epidermal physiology [55]. GH, IGF-1, IGF-2, and IGF-Bps are present in the skin and are involved in its physiological homeostasis, including the dermo-epidermal cross talking. Thus, systemic paracrine and/or autocrine cutaneous activity of the GH-IGF system contributes to skin homeostasis [55, 56]. While GH system is abated during aging, GH supplementation induces skin changes, a part of which may correspond to some corrective effects on aging skin [57, 58]. The progressive decline in DHEA serum concentration with age, and conversely its supplementation have not demonstrated prominent effects on the skin except on sebum production. Sex hormones manifest a variety of biological and immunological effects in the skin [59]. In responsive women, estrogen, alone or together with progesterone, has been reported to prevent or reverse skin atrophy, dryness, and wrinkles associated with chronological aging or photoaging. In responsive women, estrogen and progesterone stimulate proliferation of keratinocytes while estrogen suppresses apoptosis and thus prevents epidermal atrophy. Estrogen also enhances collagen synthesis, and estrogen and progesterone suppress collagenolysis by reducing MMP activity in fibroblasts, thereby maintaining skin thickness. Estrogen maintains skin moisture by
6
increasing hyaluronic acid levels in the dermis. Progesterone increases sebum excretion. Both the climacteric period around menopause and the andropause decade may negatively affect the skin [59–61]. Hormone replacement therapy (HRT) during the climacteric period helps limiting these changes [62–65]. However, there is a limitation because it seems that good and poor responders exist [66]. Smoking habit may also interfere with the treatment result [67].
Catabolic Aging The elderly are often accompanied by a substandard diet deficiency in many of the nutrients thought to be essential for maintaining health. Protein-containing foods such as meat and fish tend to be too expensive or troublesome to prepare. Dietary faddism, confusional states, and forgetfulness are also responsible for an inadequate diet. These situations predispose to skin changes that often amplify the alterations induced by age-related deficiencies. Any insufficient intake of fresh fruits and/or vegetables leads to vitamin C deficiency and to scurvy. It results in a defect in coagulation and purpura, particularly in a typical punctate perifollicular pattern on the legs. In the elderly, iron deficiency is common and may result in anemia, generalized pruritus, and diffuse hair loss. Essential fatty acid and vitamin A deficiencies due to dietary faddism or deprivation in the elderly are responsible for xerosis [68]. Many of the elderly are also deficient in zinc, and this may impair wound healing. Zinc supplementation, however, does not improve healing. Chronic hemodialysis is another model of catabolic aging affecting the mechanical properties of skin [69, 70].
Gravitational Aging Skin of any part of the body is subjected to intrinsic and extrinsic mechanical forces. Among them, earth gravitation influences skin folding and sagging during aging. Any force generated by the skin or applied to it, transduces information to cells that may in turn respond to it [71, 72]. The effects of mechanobiology are particularly evidenced in the fibroblasts, dermal dendrocytes, keratinocytes, and melanocytes [73–75]. Physical forces of gravity involve mechanotransduction in the skin [76] and affect cell tensegrity and the cell mechanosensitive ion channels. As a result, the structure of the dermal extracellular matrix is affected.
51
52
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Physiological Variations During Aging
Diversity of Wrinkles There is evidence that wrinkles are not related to the genuine microrelief [77, 78]. In addition, the microanatomical support of wrinkles is varied [77–80]. It depends on subtle changes in the structure of the superficial dermis elastotic deposits in the upper reticular dermis, loosening of the hypodermal connective tissue strands, or, inversely, on focal hypertrophic binding of the dermis to the underlying facial muscles [78, 80]. The wrinkle severity rating [81] is influenced by the nature of the altered connective tissue. Similarly, the skin mechanical properties are under these influences [82, 83]. Photoaged facial skin does not always present clinically with characteristic wrinkling. In some individuals, usually of light phototype, smooth unwrinkled skin and telangiectasia predominate. These people appear to be more at risk of developing basal cell carcinomas on sun-exposed facial skin [84, 85]. There is an apparent inverse relationship between the degree of facial wrinkling and the occurrence of facial basal cell carcinomas [85]. Mechanistically, little is known regarding how these two clinical outcomes occur in response to the same sun-exposure stimulus. Smoking effects on skin aging are probably mediated by the increased production of collagenase and elastase, which is an additional cause of wrinkling [86–88]. Degradation of elastic fibers by ROS and repeated mechanical solicitations by some muscle contractions play a putative role in the formation of the smoker’s wrinkles.
2. 3. 4. 5. 6.
7. 8. 9.
10.
11. 12. 13.
14.
15. 16.
Conclusion Aging is apparent at all levels of the physiology and anatomy of the body. Organs, tissues, cells, and molecules have their own aging processes, which differ in their clinical relevance. The individual may perceive a global appearance of skin aging. By contrast, prevention and correction of skin aging may benefit from targeting some of the specific underlying biological processes.
17. 18.
19. 20.
21.
Cross-references > The
22.
Stratum Corneum and Aging 23.
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67. Castelo-Branco C, Figueras F, Martinez de O saba MJ, Vanrell JA. Facial wrinkling in postmenopausal women effects of smoking status and hormone replacement therapy. Maturitas. 1998;29:75–86. 68. Uhoda E, Petit L, Pie´rard-Franchimont C, Pie´rard GE. Ultraviolet light-enhanced visualization of cutaneous signs of carotene and vitamin A dietary deficiency. Acta Clin Belg. 2004;59:97–101. 69. Deleixhe-Mauhin F, Pie´rard-Franchimont C, Rorive G, Pie´rard GE. Influence of chronic haemodialysis on the mechanical properties of skin. Clin Exp Dermatol. 1994;19:130–133. 70. Uhoda I, Petit L, Krzesinski JM, Pie´rard-Franchimont C, Pie´rard GE. Effect of haemodialysis on acoustic shear wave propagation in the skin. Dermatology. 2004;209:95–100. 71. Wang N, Butler JP, Ingber DE. Mechano-transduction across the cell surface and through the cytoskeleton. Science. 1993;260: 1124–1127. 72. Silver FH, Siperko M, Seehra GP. Mechanobiology of force transduction in dermal tissue. Skin Res Technol. 2003;9:3–23. 73. Ingber DE. Tensegrity: the architectural basis of cellular mechanotransduction. Annu Rev Physiol. 1997;59:575–599. 74. Hermanns-Leˆ T, Uhoda I, Pie´rard-Franchimont C, Pie´rard GE. Factor XIIIa-positive dermal dendrocytes and shear wave propagation in human skin. Eur J Clin Invest. 2002;32:847–851. 75. Quatresooz P, Hermanns JF, Paquet P, Pie´rard GE. Mechanobiology and force transduction in scars developed in darker skin types. Skin Res Technol. 2006;12:279–282. 76. Nizet JL, Pie´rard-Franchimont C, Pie´rard GE. Influence of the body posture and gravitational forces on shear wave propagation in the skin. Dermatology. 2001;2002:177–180. 77. Pie´rard GE, Uhoda I, Pie´rard-Franchimont C. From skin microrelief to wrinkles. An area ripe for investigation. J Cosmet Dermatol. 2003;2:21–28.
78. Quatresooz P, Thirion L, Pie´rard-Franchimont C, Pie´rard GE. The riddle of genuine skin microrelief and wrinkles. Int J Cosmet Sci. 2006;28:389–395. 79. Bosset S, Barre´ P, Chalon A, Kurfurst R, Bonte´ F, Andre´ P, et al. Skin ageing: clinical and histopathologic study of permanent and reducible wrinkles. Eur J Dermatol. 2002;12:247–252. 80. Pie´rard GE, Lapie`re ChM. The microanatomical basis of facial frown lines. Arch Dermatol. 1989;125:1090–1092. 81. Day DJ, Littler CM, Swift RW, Gottlieb S. The wrinkle severity rating scale: a validation study. Am J Clin Dermatol. 2004;5:49–52. 82. Hermanns-Leˆ T, Jonlet F, Scheen A, Pie´rard GE. Age-and-body mass index-related changes in cutaneous shear wave velocity. Exp Gerontol. 2001;36:363–372. 83. Hermanns-Leˆ T, Uhoda I, Smitz S, Pie´rard GE. Skin tensile properties revisited during aging. Where now, where next? J Cosmet Dermatol. 2004;3:35–40. 84. Kricker A, Armstrong BK, English DR. Sun exposure and nonmelanocytic skin cancer. Cancer Causes Control. 1994;5:367–392. 85. Brooke RCC, Newbold SA, Telfer NR, Griffiths CE. Discordance between facial wrinkling and the presence of basal cell carcinoma. Arch Dermatol. 2001;137:751–754. 86. Ernster VL, Grady D, Miike R, Back D, Selby J, Kerlikowske K. Facial wrinkling in men and women by smoking status. Am J Public Health. 1995;85:78–82. 87. Koh JS. Cigarette smoking associated with premature facial wrinkling: image analysis of facial skin replicas. Int J Dermatol. 2002;41:21–27. 88. Raitio A, Tuomas H, Kokkonen N, Salo T, Sorsa T, Hanemaaijer R, et al. Levels of matrix metalloproteinase-2, -9 and -8 in the skin, serum and saliva of smokers and non-smokers. Arch Dermatol Res. 2005;297:242–248.
52 Pigmentation in Ethnic Groups Rupa Pugashetti . Howard I. Maibach
Introduction
Mechanism of Pigmentation
In order to help understand the pigmentary changes which occur throughout the aging process and in the management of aging skin, it is necessary to examine how pigmentation varies among different ethnicities. Skin color among different ethnicities has important social and political connotations, both historically and at the present time. While it is clear that human skin is an important external feature which can distinguish people of different races, less clear is how variations in human skin pigmentation may contribute to differences in skin structure, function, and pathophysiology. As the demographics of the United States change, the US Census Bureau estimates that by 2050, approximately half of the US resident population will comprise individuals of color [1]. The question of associating race and pigmentation is repeatedly raised, and a recent study demonstrated the need for caution when using pigmentation as a proxy for race or genetic ancestry [2]. Nonetheless, as dermatologists treat more patients of varying ethnic backgrounds and with diverse skin types, an understanding of differences in pigmentation and skin structure and function becomes more important.
Human skin color is determined primarily by melanin synthesis. Melanin can act as an effective sunscreen, protecting darker-pigmented individuals from burning and development of skin cancer [3]. It is now well established that heavy pigmentation in areas of high solar radiation helps protect the skin from carcinogenic effects of ultraviolet light [4]. This might have played a role in selection of darker skin colors evolutionarily in African populations, but how lighter skin was produced as early humans migrated out of Africa and moved into Europe still remains controversial. Additionally, there is a correlation between latitude and skin color; skin is lighter in regions towards the north. This correlates with ultraviolet radiation incidence, which is higher near the equator and lower at higher latitudes. In human skin pigmentation, constitutive skin color refers to genetically determined levels of melanin pigmentation without exposure to ultraviolet radiation or other environmental influences, while facultative skin color refers to increases in melanin pigmentation above the constitutive level induced by exposure to ultraviolet light [5]. Human skin color is dominated by both hemoglobin, which provides red color via the network of capillaries in the skin, and melanin, which provides various gradations of brown coloring to the skin surface. There does not appear to be any significant difference in hemoglobin content among various ethnicities, and there is a constant vascular supply under conditions of constant activity and temperature [6]. Furthermore, the effect of hemoglobin in blood flowing in the dermal papillary layer is easily masked by pigments present in the overlying epidermal layers. Thus, it is primarily differences in the amount of melanin which are responsible for normal variation in human skin pigmentation. Melanin synthesis takes place inside melanosomes, organelles present in the melanocyte. Melanin pigment granules are subsequently transferred to keratinocytes [7]. Specifically, melanosomes are trafficked along dendrites; they are moved along microtubules before being captured
Methods The EMBASE database was searched using the terms ‘‘race,’’ ‘‘race difference,’’ ‘‘minority group,’’ ‘‘ethnicity,’’ ‘‘ethnic difference,’’ ‘‘ethnic or racial aspects,’’ and ‘‘skin pigmentation’’ in different combinations. Altogether, 165 articles were retrieved, and abstracts were subsequently reviewed for relevance to the topic. The PubMed database was searched with the specific terms ‘‘minority groups,’’ ‘‘population groups,’’ and ‘‘skin pigmentation’’ in different combinations. Altogether, 295 results were retrieved, and abstracts were reviewed for relevance to the topic. Editorial articles, population-based studies, basic science, and clinical studies were included provided the subject matter was relevant to the topic discussed.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_52, # Springer-Verlag Berlin Heidelberg 2010
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at dendrite tips through a molecular complex which includes myosin [8]. Melanosomes are pinched off or released from the melanocyte dendrite and subsequently phagocytosed by adjacent keratinocytes. Suprabasal keratinocytes move upwards through epidermal keratinocyte layers where melanocyte dendrites continuously transfer melanosomes to maturing keratinocytes. Eventually, melanin is shed from top layers of stratum corneum with the cornified keratinocytes. The proteinase activated receptor-2 (PAR-2) is a receptor expressed in keratinocytes, and is thought to play a fundamental role in this process of melanosome transfer and skin pigmentation [9]. The overall regulation of melanin formation is complex, and numerous factors affect mammalian melanocytes to regulate melanin synthesis; these include melanotropin, estrogen and progesterone, and agouti signal protein [10]. Further studies must elucidate the complex regulation of melanin formation, the regulation of phagocytosis of melanosomes by keratinocytes, and how signaling intermediates stimulate melanosome transfer. Additionally, it is not entirely clear whether melanosomes are transferred from melanocytes to keratinocytes in membrane-bound clusters or as individual melanosomes, or both.
Variations in Pigmentation and Ultrastructural Differences in Skin Montagna and Carlisle examined the morphology of black and white facial skin among adult women 22–50 years of age [11]. When comparing the epidermis of black skin to white skin, the epidermis of black skin demonstrated more and larger singly distributed melanosomes in corneocytes and keratinocytes. Additionally, the epidermal stratum lucidum was not altered by sunlight exposure in black skin whereas the stratum lucidum was usually distorted on exposure to the sun in white skin. White skin demonstrated frequent areas of atrophy, while only 1 of 19 black women in the study demonstrated atrophic spots in the epidermis. One of the key indices of dermal photodamage is the presence of elastotic material. Black skin demonstrated minimal elastosis while white skin showed variable amounts of moderate to extensive elastosis. The authors speculated that the greater numbers of melanosomes and distribution in black skin perhaps protect the epidermis from photodamage. Additionally, the dermis of black skin demonstrated more fiber fragments made of collagen fibrils and glycoproteins, as well as numerous and larger fibroblasts. Finally, black skin showed more mixed apocrine-eccrine sweat glands, as well as more blood and lymphatic vessels when compared to white skin.
Overall, the most striking difference demonstrated between black and white skin appears to be the size of melanosomes and distribution pattern of melanosomes. In darkly pigmented skin, large melanosomes are surrounded by the membrane whereas smaller melanosomes are grouped or clustered together in a single membrane in lighter skin [12]. Additionally, melanosomal packaging is closer to the basal layer in more darkly pigmented skin as compared to Caucasian skin [13]. Regarding variations in human skin color, the density of pigment producing melanocytes in the skin (approximately 1,000/mm2) has not been known to vary with ethnicity [14]. Conversely, the ratio between eumelanin and pheomelanin synthesis was demonstrated to be higher in black compared to white skin [15]. Thong et al. examined the patterns of melanosome distribution in keratinocytes of Asian skin and compared this to light Caucasian skin and dark African-American skin [16]. In this study, the distribution pattern of melanosomes transferred to keratinocytes in the photoprotected skin (volar forearm) from normal Asian individuals was examined. Results demonstrated that melanosomes in keratinocytes of Asian skin are distributed as both individual and clustered melanosomes, with 62.6% individual and 37.4% clustered. In dark skin keratinocytes, melanosomes are predominantly individual (88.9%) and in light Caucasian skin keratinocytes melanosomes are predominantly clustered (84.5%). Thus, the melanosome distribution in Asian keratinocytes seems to be intermediate between light Caucasian and dark keratinocytes. When examining the size of melanosomes, there appeared to be a variation in size with ethnicity; melanosomes in dark skin were the largest, followed by melanosomes of Asian and then Caucasian skin. Furthermore, melanosomes which are distributed individually tend to be larger as compared to clustered melanosomes. Minwalla et al. examined how keratinocytes play a role in regulating the distribution patterns of recipient melanosomes in vitro [17]. Cocultures using melanocytes and keratinocytes from different racial backgrounds were studied with electron microscopy. When keratinocytes from dark skin were cocultured with melanocytes from either dark or light skin, recipient melanosomes were predominantly individual as opposed to clustered. However, when keratinocytes from light skin were cocultured with melanocytes from dark or light skin, recipient melanosomes were predominantly clustered as opposed to individual. Thus, recipient melanosomes overall are predominantly distributed in membrane-bound clusters from light skin keratinocytes, and distributed individually by dark skin keratinocytes. Furthermore, melanosome
Pigmentation in Ethnic Groups
size was not related to how melanosomes were distributed. Authors suggested that keratinocyte regulatory factors may determine how exactly recipient melanosomes are distributed. Melanin content in photoexposed and photoprotected skin has been examined among varying ethnicities including African, Indian, Mexican, Chinese, and European skin [18]. The lightly pigmented skin types had approximately half as much epidermal melanin as compared to more darkly pigmented skin types. Furthermore, the melanin composition among the lighter skin types (Mexican, Chinese, and European skin) was more enriched with lightly colored, alkali-soluble melanin pigments such as pheomelanin and eumelanin. Epidermal melanin content is greater in chronically photoexposed skin as compared to photoprotected skin, regardless of ethnic background. This analysis, like previous studies, also demonstrated that melanosome size varies with ethnicity: African skin had the largest melanosomes, followed by Indian, Mexican, Chinese, and finally European skin. Thus, the amount of melanin, composition of melanin, and differences in melanosome size may all play roles in determining skin pigmentation. Halprin et al. reported that glutathione may play a role in the genetically determined differences in skin color among different races [19]. This sulfydryl-containing epidermal compound plays a role in melanin formation. Halprin described that the tripeptide glutathione (g-glutamyl-cysteinyl-glycine) is present in the human epidermis in sufficient concentrations to be the inhibitor of melanin formation from tyrosine by tyrosinase. Overall, reduced glutathione and the enzyme glutathione reductase, which is needed to maintain glutathione in the reduced state, are found in lower concentrations in African epidermal skin as compared to Caucasian epidermis.
The Role of Tyrosinase Iwata et al. examined the relationship between tyrosinase activity and skin color in human foreskins [20]. Darker skin types appear to have a higher level of melanin production due to the constitutively higher level of activity of tyrosinase, the rate limiting enzyme in melanin synthesis. Tyrosinase activity was measured with two separate assays, a tyrosinase hydroxylase assay and a [14C] melanin assay, measuring both the hydroxylation of tyrosine to dopa and the conversion of [14C] tyrosine to [14C] melanin. In black foreskin homogenates, tyrosinase activity was measured at nearly three times the activity compared to white skin samples. Tyrosinase activity was generally
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correlated with melanin content in the skin. Variations in tyrosinase activity may be due to different amounts of enzyme, but also potentially due to differences in catalytic activity of the enzyme in melanocytes. Tyrosinase is the rate-limiting enzyme in melanin synthesis, and its overall activity is higher in melanocytes of black skin as compared to Caucasian skin [21]. Fuller et al. examined the regulation of tyrosinase in black and Caucasian human melanocyte cell cultures. Their studies showed that variation in enzyme activity is due to differences in the catalytic activity of preexisting tyrosinase rather than differences in tyrosinase abundance or gene activity. In the melanosomes of black melanocytes, tyrosinase has high catalytic activity while in Caucasian melanocytes, the melanosome-bound enzyme is largely inactive. Furthermore, staining of Caucasian melanocytes with a weak base demonstrated that Caucasian melanosomes are acidic organelles as compared to more neutral in darker skin. Thus, differences in melanosome pH may contribute to variations in pigmentation. Moreover, tyrosinase is inactive in an acidic environment, so it is largely inactive in Caucasian melanosomes as compared to higher activity in melanosomes of black skin. Maeda et al. compared melanogenesis in human black and light brown melanocytes [22]. Melanin pigment in both human black and light brown melanocytes contains eumelanin and pheomelanin, with black melanocytes containing a larger amount. Tyrosinase activity was higher in the black melanocytes as compared to light brown melanocytes. The differences in pigmentation of the two human melanocyte cell lines (black and light brown) seemed to be derived from differences in activity of tyrosinase, as well as other specific proteins affecting the constitution of melanin polymers. These other proteins include tyrosinase-related protein-1 and dopachrome tautomerase, which modulate distal steps in melanogenesis.
Genetics of Skin Pigmentation Only recently have investigators begun to develop a better understanding of the complex genetic basis for normal variation in human skin pigmentation. Much of the research investigating the genes playing a role in melanin synthesis have been carried out in animal models. With the help of mouse coat color mutations, many of the biochemical pathways involved in melanin synthesis have been studied and elucidated; over 100 genes have been identified which can affect mouse coat color [23]. Many of these genes have corresponding human phenotypes. One of the genes affecting normal variation in skin
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pigmentation is the melanocortin 1 receptor (MC1R) gene. Mutations in this gene affect pigmentation in humans, mice, cattle, horses, sheep, pigs, and chickens among other animals. Specifically, this gene product is in the melanocyte cell membrane, and is the receptor for a-melanocyte stimulating hormone. While polymorphisms in MC1R may play a vital role in shaping human pigmentation, this process is complex and multiple genes likely determine normal variation in skin pigmentation [24]. In addition, some variation in human skin color is associated with variation in TYR and OCA2, two of the known pigmentation genes [25]. More recent studies have focused on SLC24A5, a putative cation exchanger, which was originally studied in zebrafish and is suggested to play a key role in human skin pigmentation [26]. The SLC24A5 exchanger localizes to an intracellular membrane, likely the melanosome or its precursor; it is thought that variations in this gene may help explain some of the difference in pigmentation between European-Americans and AfricanAmericans. Studies have shown that the genes MATP, TYR, and SLC24A5 may play a predominant role in the evolution of lighter skin in Europeans but not East Asians, suggesting that there is a recent convergent evolution of lighter skin pigmentation in East Asians and Europeans [27]. Interestingly, such data also suggest that European skin turned lighter approximately 6,000–12,000 years ago, contradicting a previous hypothesis that European skin grew more pale approximately 40,000 years ago [28].
Response to Ultraviolet Radiation Tadokoro et al. examined the mechanism of skin tanning in different ethnic groups [29]. The effect of ultraviolet radiation after one minimal erythemal dose exposure was studied. Overall, the density of melanocytes present at the epidermal–dermal junction did not change significantly 1 week following ultraviolet light exposure, and this density was similar among different ethnic skin types. However, the distribution of melanin from the lower layers to middle layers of the skin epidermis was more dramatic in darker skin as compared to lighter skin following ultraviolet exposure. Erythema responses have also been examined in patients of different complexions [30]. The minimal erythema dose (MED), defined as the smallest quantity of radiation needed to produce a barely perceptible erythema, was determined in Caucasians and in differing complexions of African-American skin. Among light, medium, and dark-complexioned African-Americans, no minimal
erythema response was typical. Instead, a spectrum of responses was found which was directly proportional to the degree of pigmentation. Additionally, the average MED of the darker-complexioned African-Americans was 33 times greater than that of Caucasians. Caucasian skin had the smallest amount of pigment and smallest melanosomes, which were mostly contained within melanosome complexes. Further findings included that melanosome size is directly proportional to the intensity of skin pigmentation, and darkly pigmented subjects have larger, wider and denser melanosomes. Generally, with increasing pigmentation, the size of melanosomes, the proportion of singly dispersed melanosomes, and the MED were all shown to increase. Authors suggested that the increased resistance of darker skin to the damaging effects of ultraviolet radiation may be due to larger, more light-absorbing, individually dispersed melanosomes. Furthermore, melanosomes in darkly pigmented skin are degraded less by lysosomes, resulting in more light-absorbing bodies in the stratum corneum [31].
Skin Structure and Function The skin barrier, made primarily of terminally differentiated keratinocytes in a lipid matrix composed of fatty acids, ceramides, and cholesterols, helps to determine the skin’s integrity [32]. When examining the skin barrier and its components, darkly pigmented skin does appear to have some inherent structural and functional differences as compared to lighter skin [33]. One study compared transepidermal water loss and water content among black, white, Latino, and Asian populations [34]. The transepidermal water loss measurements were highest in black, followed by white, Latino, and Asian populations in decreasing order. Furthermore, stratum corneum lipids were significantly lower in black epidermal layers as compared to other races. The skin barrier of more darkly pigmented skin is thought to be more resistant to injury and recover more quickly from injury [35]. Marshall et al. demonstrated that black skin may have decreased susceptibility to cutaneous irritants, suggesting that the black skin barrier may be stronger [36]. Another study by Weigand et al. demonstrated that there may be greater numbers of stratum corneum cell layers in black skin and possibly greater cell cohesiveness as well [37]. However, it has also been suggested that darkly pigmented persons are more susceptible to cold injury as compared to those with lighter pigmentation [38]. One concern about varying levels of pigmentation among human skin of different ethnic backgrounds is
Pigmentation in Ethnic Groups
the possible effect on cutaneous synthesis of vitamin D. Matsuoka et al. examined this further by testing serum vitamin D levels and levels of active serum metabolites among white, East Asian, South Asian, and black subjects following a fixed dose of ultraviolet B radiation [39]. Because the amount of epidermal melanin determines the number of photons, which reach the lower epidermal layers where vitamin D3 synthesis takes place, vitamin D formation could be affected by individual characteristics of melanization. However, when assessing vitamin D nutritional status by measuring serum 25-hydroxyvitamin D, ethnic background had only a marginal effect with higher levels in whites compared to blacks. Regarding its active serum metabolite 1, 25-dihydroxyvitamin D, levels were similar across all groups. Authors concluded that varying levels of pigmentation do not prevent the generation of normal levels of active vitamin D metabolites, while increasing pigmentation in the epidermis still exerts a strong photoprotective effect.
Objective Measurements of Skin Color Advances have been made in pigmentation measurement devices, allowing for easy measurement of epidermal melanin using tristimulus reflectometry, narrow-band spectroscopy, and diffuse reflectance spectroscopy. The tristimulus chromameter utilizes the L*a*b* color system to determine skin color: L* represents skin reflectance or lightness, a* measures color saturation from red to green, and b* measures color saturation from yellow to blue. The aforementioned measurement devices have been utilized in objectively measuring human skin pigmentation and in examining the impact of melanin on human skin color. Lee et al. evaluated the Minolta CR-400 chromameter (Tokyo, Japan) as an objective measurement of periocular and facial pigmentation in subjects form different ethnic backgrounds [40]. African-American, Caucasian, and Hispanic subjects had facial and periocular skin color measurements performed. Using the L*a*b* color system, significant differences in L* were observed among all ethnic groups, while a* and b* were less sensitive to pigmentation differences. Additionally, the value L* (i.e. skin reflectance or lightness) demonstrated significant differences between different Fitzpatrick skin types III–VI, the more heavily pigmented groups. The Minolta CR-400 chromameter reliably measures facial pigmentation, and can be utilized when evaluating changes in skin pigmentation in studies; the chromameter showed good inter- and intra-instrument reliability as well.
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Shriver and Parra compared two methods to measure pigmentation in skin and hair in a group of subjects including European-Americans, African-Americans, South Asians, and East Asians [41]. The tristimulus colorimeter Photovolt ColorWalk (Indianapolis, IN) which uses the L*a*b* color system was compared to the DermaSpectrometer narrow-band reflectometer (Hadsund, Denmark) which measures pigment in terms of erythema and melanin indices. Both types of instruments did provide accurate estimates of pigment level in the skin. However, measurements performed by the narrow-band reflectometer were less affected by the greater redness of specific body sites due to increased vascularization. Alaluf et al. examined the impact of epidermal melanin on objective measurements of human skin color [42]. The tristimulus chromameter was used to obtain measurements of human skin color in different ethnic skin types. Tristimulus L*a*b* measurements were made in European, Mexican, Chinese, Indian, and African subjects. Overall, darker skin types tend to have lower L* values, higher a* values and higher b* values when compared to constitutively lighter skin types. Results demonstrated that total epidermal melanin is the primary determinant of L* values (i.e., skin reflectance or lightness). Melanosome size also has a significant influence on L* values and larger melanosomes are associated with a darker skin color, as discussed previously. Based on the strength of correlations observed in this study, epidermal melanin content still seems to play a greater role than melanosome size in determining skin color.
Conclusion Many advances have been made in understanding the genetic, molecular, and cellular differences underlying normal variation in human skin pigmentation. However, further studies must be carried out in order to investigate the complex genetic pathway underlying melanin synthesis, the role of genetic variation in epidermal pigmentation, and to elucidate differences in skin pathophysiology among humans from different ethnic backgrounds. As knowledge develops about the intricate process of pigmentation and distinctions between ethnic groups, it can be further understood how pigmentary changes occur with aging, and consequently develop more effective management.
Cross-references > Hyperpigmentation > The
in Aging Skin New Face of Pigmentation and Aging
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References 1. US Census Bureau. Interim projections by age, sex, race, and Hispanic origin, 2004. Available at: http://www.census.gov/ipc/www/ userinterimproj/. Accessed September 22, 2004. 2. Parra EJ, Kittles RA, Shriver MD. Implications of correlations between skin color and genetic ancestry for biomedical research. Nat Genet. 2004;36:S54–60. 3. Jackson IJ. Identifying the genes causing human diversity. Eur J Hum Gen. 2006;14:978–980. 4. Harrison GA. Differences in human pigmentation: measurement, geographic variation, and causes. J Invest Dermatol. 1973;60: 418–426. 5. Quevedo WC Jr., Fitzpatrick TB, Pathak MA, Jimbow K. Role of light in human skin color variation. Am J Phys Anthrop. 1975;43: 393–408. 6. Kalla AK. Human skin pigmentation, its genetics and variation. Humangenetik. 1974;21:289–300. 7. Westerhof W. A few more grains of melanin. Int J of Dermatol. 1997;36:573–574. 8. Scott GA. Melanosome trafficking and transfer. In: Nordlund JJ, Boissy RE, Hearing VJ, King RA, Oetting WS, Ortonne JP (eds) The Pigmentary System, Physiology and Pathophysiology, 2nd ed. Massachusetts: Blackwell Publishing Ltd, 2006, pp. 171–180. 9. Seiberg M. Keratinocyte-melanocyte interactions during melanosome transfer. Pigment Cell Res. 2001;14:236–242. 10. Hearing VJ. The regulation of melanin formation. In: Nordlund JJ, Boissy RE, Hearing VJ, King RA, Oetting WS, Ortonne JP (eds) The Pigmentary System, Physiology and Pathophysiology, 2nd ed. Massachusetts: Blackwell Publishing Ltd, 2006, pp. 191–212. 11. Montagna W, Carlisle K. The architecture of black and white facial skin. J Am Acad Dermatol. 1991;24:929–937. 12. Szabo G, Gerald AB, Pathak MA. Racial differences in human pigmentation on the ultrastructural level. J Cell Biol. 1968;39: 132a–133a. 13. Toda K, Pathak MA, Parrish JA, Fitzpatrick TB, Quevedo WC Jr. Alteration of racial differences in melanosome distribution in human epidermis after exposure to ultraviolet light. Nat New Biol. 1972;236:143–145. 14. Szabo G. The number of melanocytes in human epidermis. Br Med J. 1954;1:1016–1017. 15. Thody AJ, Burchill SA, Ito S. Epidermal eumelanin and phaeomelanin concentrations in different skin types and in response to PUVA. Br J Dermatol. 1990;123:842–845. 16. Thong HY, Jee SH, Sun CC, Boissy RE. The patterns of melanosome distribution in keratinocytes of human skin as one determining factor of skin colour. Br J Dermatol. 2003;149:498–505. 17. Minwalla L, Zhao Y, Le Poole IC, Wicket RR, Boissy RE. J Invest Dermatol. 2001;117:341–347. 18. Alaluf S, Atkins D, Barrett K, Blount M, Carter N, Heath A. Ethnic variation in melanin content and composition in photoexposed and photoprotected human skin. Pigment Cell Res. 2002;15:112–118. 19. Halprin KM, Ohkawara A. Glutathione and human pigmentation. Arch Derm. 1966;94:355–357. 20. Iwata M, Corn T, Iwata S, Everett MA, Fuller BB. The relationship between tyrosinase activity and skin colour in human foreskins. J Invest Dermatol. 1990;95:9–15. 21. Fuller BB, Spaulding DT, Smith DR. Regulation of the catalytic activity of preexisting tyrosinase in black and caucasian human melanocyte cell cultures. Exp Cell Res. 2001;262:197–208.
22. Maeda K, Yokokawa Y, Hatao M, Naganuma M, Tomita Y. Comparison of the melanogenesis in human black and light brown melanocytes. J Dermatol Sci. 1997;14:19–206. 23. Westerhof W. Evolutionary, biologic, and social aspects of skin color. Dermatol Clin. 2007;25:293–302. 24. Makova K, Norton H. Worldwide polymorphism at the MC1R locus and normal pigmentation variation in humans. Peptides. 2005;26:1901–1908. 25. Shriver MD, Parra EJ, Dios S, Bonilla C, Norton H, Jovel C, et al. Skin pigmentation, biogeographical ancestry and admixture mapping. Hum Genet. 2003;112:387–399. 26. Lamason RL, Mohideen MPK, Mest JR, Wong AC, Norton HL, Aros MC, et al. SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans. Science. 2005;310:1782–1786. 27. Norton HL, Kittles RA, Parra E, McKeigue P, Mao X, Cheng K, et al. Genetic evidence for the convergent evolution of light skin in Europeans and East Asians. Mol Biol Evol. 2007;24:710–722. 28. Gibbons A. American Association of physical anthropologists meeting. European skin turned pale only recently, gene suggests. Science. 2007;316:364. 29. Tadokoro T, Yamaguchi Y, Batzer J, Coelho SG, Zmudzka BZ, Miller SA, et al. Mechanisms of skin tanning in different racial/ ethnic groups in response to ultraviolet radiation. J Invest Dermatol. 2005;124:1326–1332. 30. Olson RL, Gaylor J, Everett MA. Skin color, melanin, and erythema. Arch Dermatol. 1973;108:541–544. 31. Olson RL, Nordquist J, Everett MA. The role of lysosomes in melanin physiology. Br J Dermatol. 1970;83:189–199. 32. Baumann L, Rodriguez D, Taylor SC, Wu J. Natural considerations for skin of color. Cutis. 2006;78(6):2–20. 33. Taylor S, Woolery-Lloyd H. Pigmentation disorders in skin of color: the role of natural substances. Semin Cutan Med Surg. 2008;27:14–15. 34. Sugino K, Imokawa G, Maibach H. Ethnic difference of stratum corneum lipid in relation to stratum corneum function [abstract]. J Invest Dermatol. 1993;100:597. 35. Reed JT, Ghadially R, Elias PM. Effect of race, gender, and skin type of epidermal permeability barrier function. J Invest Dermatol. 1994;102:537. Abstract. 36. Marshall E, Lynch V, Smith H. Variation in the susceptibility of the skin to dichloroethylsulfide. J Pharmacol Exp Ther. 1919;12: 291–301. 37. Weigand D, Haygood C, Gaylor J. Cell layers and density of negro and caucasian stratum corneum. J Invest Dermatol. 1974;62: 563–568. 38. Post PW, Farrington D Jr., Binford RT Jr. Cold injury and the evolution of ‘‘white’’ skin. Human Biology. 1975;47:65–80. 39. Matsuoka LY, Wortsman J, Haddad JG, Kolm P, Hollis BW. Racial pigmentation and the cutaneous synthesis of vitamin D. Arch Dermatol. 1991;127:536–538. 40. Lee JA, Osmanovic S, Viana MAG, Kapur R, Meghpara B, Edward DP. Objective measurement of periocular pigmentation. Photodermatol Photoimmunol Photomed. 2008;24:285–290. 41. Shriver MD, Parra EJ. Comparison of narrow-band reflectance spectroscopy and tristimulus colorimetry for measurements of skin and hair color in persons of different biological ancestry. Am J Phys Anthropol. 2000;112:17–27. 42. Alaluf S, Atkins D, Barrett K, Blount M, Carter N, Heath A. The impact of epidermal melanin on objective measurements of human skin colour. Pigment Cell Res. 2002;15:119–126.
12 Possible Involvement of Basement Membrane Damage by Matrix Metalloproteinases and Serine Proteinases in Skin Aging Process Satoshi Amano
Introduction Skin aging can be classified into two types: intrinsic aging and photoaging [1]. Intrinsic aging is the basic biological process common to all living things and is characterized as an age-dependent deterioration of skin functions and structures, such as epidermal atrophy and epidermal– dermal junctional flattening [2]. Photoaging is well known to be a consequence of chronic exposure of the skin to sunlight. Sun-exposed skin, such as face or neck skin, clearly appears to be ‘‘prematurely aged’’ in comparison with the relatively sun-protected skin of the trunk or thigh, and is characterized by various clinical features, including wrinkles, sagging, roughness, sallowness, pigmentary changes, telangiectasis, and neoplasia [3, 4]. The histological features of sun-exposed skin include cellular atypia, loss of polarity, flattening of the dermal–epidermal junctions (DEJ), a decrease in collagen, and dermal elastosis [2, 5]. The basement membrane (BM) at the DEJ has many functions, of which the most obvious is to tightly link the epidermis to the dermis [6]. It also determines the polarity of the epidermis and provides a barrier against epidermal migration. Once the BM has been assembled, the epidermal cells recognize the surface adjacent to the BM as the basal surface. Stratification of the epidermis proceeds with the proliferating cells remaining attached to the BM and the daughter cells migrating into the upper layers [7–9]. It is thought that the BM influences epidermal differentiation and maintains the proliferative state of the basal layer. Under normal circumstances, the BM prevents direct contact of epidermal cells with the dermis. Another important function of the BM derives from the positioning of the structure between the epidermal and the dermal cells. The epidermis and the dermis do not function independently [10]. Instead, normal skin
homeostasis requires the constant passage of signals back and forth between the two cell types. In general, these signals are small molecules, synthesized in one compartment, that diffuse to the other compartment. In other words, these signals must cross the BM. Components of the BM can selectively facilitate or prevent the passage of these signals. In some cases, the signaling molecules are stored by the BM and released only if the BM is damaged or destroyed. Thus, epidermal–dermal communication through the BM is extremely important. The BM may be divided into three layers on the basis of morphological studies: the lamina lucida, the lamina densa, and the lamina fibroreticularis [11], as shown in > Fig. 12.1. Lamina densa is a sheet-like structure, which is mainly composed of type IV collagen. Lamina lucida is a region between the lamina densa and the epithelia, forming electron-dense plaques, hemidesmosomes, which mainly consist of a6b4integrin and the bullous pemphigoid antigen 2 (180 kDa). The BM contains unique structures that maintain the attachment of the epidermis. The components of the attachment complex provide links to the intracellular intermediate filament network of basal keratinocytes and to the extracellular matrix of the papillary dermis. One of the key components of the anchoring complex is laminin 5 (332). Past work has shown that laminin 332 is essential to epidermal attachment, as mutations in the genes encoding the laminin 332 chains underlie the severe blistering phenotype of Herlitz’ junctional epidermolysis bullosa [12]. Laminin 332 is processed extracellularly to a mature form at the a3 and g2 chains by BMP-1 and other enzymes [13–15]. It is clear that laminin 332 constitutes the anchoring filaments and binds the transmembrane hemidesmosomal integrin a6b4, which is known to be the receptor of laminin 332. With regard to binding of laminin 332 with other components of the BM or of the papillary dermis, it
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_12, # Springer-Verlag Berlin Heidelberg 2010
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. Figure 12.1 Basement membrane structure at DEJ and damaged BM structures. In the top row, the DEJ is visualized at stepwise magnification in transmission electron microscope scale. Hemidesmosome (HD), anchoring filament (af), lamina densa, and anchoring fibrils (Af) are observed and form the special anchoring complex for the attachment of the epidermis to the dermis. The lower pictures are transmission electron microscopic images of the DEJ of human skin. Disruption and reduplication of the lamina densa can be seen at the DEJ in the sun-exposed cheek skin of a 30-year-old female, while neither duplication nor disruption can be observed in the sun-protected abdomen skin of a 34-year-old female
has recently been elucidated that (1) laminin 332 directly binds type VII collagen, which forms the anchoring fibrils that insert into the papillary dermis, and (2) laminin 332 forms a covalent complex with laminin 311 or 321, and this laminin 332–311/321 complex interacts with type IV collagen in the BM through nidogen. The matrix metalloproteinases (MMPs) are zincdependent endopeptidases, and they are involved in remodeling of the extracellular matrix and also play important roles in morphogenesis, angiogenesis, arthritis, skin ulcer, tumor invasion, and metastasis [16]. Five families of MMPs have been recognized: collagenases, gelatinases, stromelysins, matrilysins, and membrane-type MMPs. These
enzymes are composed of several domains, including propeptide, catalytic, and hemopexin (except for matrilysin) domains. They are involved in the degradation of collagens, proteoglycans, and various glycoproteins [16]. Among them, gelatinase A (MMP-2 or 72 kDa type IV collagenase) and gelatinase B (MMP-9 or 92 kDa type IV collagenase) digest type IV and VII collagens, while stromelysins (MMP-3, MMP-10) degrade laminins of the BM [17]. MMPs are secreted as inactive zymogens (proMMPs) and activation of proMMPs (to active MMPs) is a prerequisite for function. Stimulation or repression of proMMP synthesis is mostly regulated at the transcriptional level by growth factors or cytokines [17]. Furthermore,
Possible Involvement of Basement Membrane Damage by Matrix Metalloproteinases and Serine Proteinases
posttranscriptional regulation of MMPs activity is controlled by tissue inhibitors of metalloproteinases (TIMPs), of which TIMP-1, TIMP-2, TIMP-3, and TIMP-4 have been characterized [18]. MMP-2 binds specifically to TIMP-2, whereas MMP-9 binds to TIMP-1 [19]. MMP-2 is constitutively expressed in many cells, including dermal fibroblasts, and has a ubiquitous tissue distribution. ProMMP-2 is activated at the cell surface by a membrane-type MMP known as MT1-MMP [20]. Plasminogen activators (PAs)/plasmin represent one of the most potent and widely expressed systems for extracellular proteolysis [21]. PAs can be produced by many cell types, including human epidermal keratinocytes [22], and they convert the widely distributed zymogen, plasminogen, to plasmin, which degrades most extracellular proteins either directly or by activating other proteases [22, 23]. Tissue-type PA (tPA) and urokinase-type PA (uPA) are the products of distinct but related genes with different patterns of expression and regulation. Physiologically, tPA is predominantly responsible for fibrinolysis, while uPA appears to be involved in pericellular proteolysis by binding to cell surfaces through a specific, high-affinity, glycosylphosphatidylinositol-anchored plasma membrane receptor [24]. Binding increases the catalytic efficiency and targets generation of plasmin to the immediate pericellular space. In skin, urokinase-type plasminogen activator activity was found to be present in the stratum corneum, as well as the basal layer after barrier disruption [25, 26]. The plasminogen/plasmin system in the epidermis is thought to be the major protease activity involved in the delay of barrier recovery [26]. The first skin-equivalent model was developed in 1981 by Bell et al. [27], who plated human keratinocytes on top of contracted collagen gel containing dermal fibroblasts. It is useful for grafting on wounds [27] or burned skin [27], to explore the dynamics of the BM [28, 29], and for studies of epidermal differentiation, dermal–epidermal interaction, tumor cell invasion, and so on [28, 30, 31].
Ultrastructural Alteration of Epidermal BM in Sun-Exposed Skin At the DEJ of sun-exposed skin, duplication of lamina densa was reported in both aged adults [2] and mouse [32], and these changes may result in a more fragile epidermal–dermal interface and weaker resistance of the epidermis to shearing forces in aged skin. It was observed that,
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even in cheek (sun-exposed) skin of a 30-year-old female, severe disruption and reduplication of lamina densa were frequently observed beneath keratinocytes and anchoring fibrils were also associated with detached lamina densa, mainly on the dermis side (> Fig. 12.1). On the other hand, in young, sun-protected skin, such as abdominal skin of a 34-year-old female, scarcely any alteration of the epidermal BM structure was apparent at the DEJ (> Fig. 12.1). In old sun-exposed skin from cheek of a 60-year-old female, the layer number of reduplicated lamina densa was increased and laminae densae branched in various directions. In contrast, skin from upper thigh of an 83-year-old female showed very little disruption or reduplication at the DEJ. However, following injury that penetrates or disrupts the BM, the epidermal cells lose contact with the BM, and come in contact with naked dermis. Under these conditions, the epidermal cells modify their behavior to cover and close the wound. Such behavior changes include the upregulation of proteolytic enzymes and other changes that accompany conversion to a migratory phenotype [33, 34].
Involvement of Matrix Metalloproteinases in Damage to Epidermal BM in Sun-Exposed Skin MMPs are considered to be involved in photoaging, since MMPs-1, 2, 3, and 9 were found to be increased by ultraviolet irradiation in experiments using human fibroblasts [28, 35–38] and human skin [39, 40]. In particular, Fisher et al. demonstrated an increase of MMPs in human skin following exposure even to an extremely low level of UVB [40], and suggested that MMPs are UV-induced aging factors. In fact, gelatinase activities have been detected in the epidermis of forehead skin by in situ gelatin zymography [41]. For further study, the skin equivalent was selected as a model for BM damage, which partially mimics the photoaging process because of missing BM structure at the DEJ and the presence of large amounts of MMPs, including gelatinases (MMP-2 and MMP-9), in the culture medium as shown in > Fig. 12.2 [28, 35]. An MMP inhibitor, CGS27023A, enhances the assembly of BM at the DEJ in the skin equivalent (> Fig. 12.3) [28, 35], suggesting that MMPs play an important role in the degradation of BM components and induce BM structural damage, such as detachment of BM from basal keratinocytes and disruption of lamina densa, which are observed in sun-exposed skin (> Fig. 12.1).
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. Figure 12.2 Skin-equivalent model. Human skin-equivalent models are prepared by using human fibroblasts and keratinocytes. On the top of contracted collagen gel containing fibroblasts, keratinocytes are plated and the next day, the keratinocyte layer is exposed to air. Multilayered epidermis with cornified layer, like normal human skin, is formed within a week on the top. However, no lamina densa structure is observed at the DEJ. The activities of MMP-2 and MMP-9 are detected in the conditioned medium by gelatin zymography
. Figure 12.3 Protective effects of MMP inhibitors against BM damage at the DEJ. Skin equivalents were cultured from day 7 through day 14 with or without a synthetic MMP inhibitor, CGS27023A. Each sample was processed for electron microscopy on day 14. Lamina densa (arrows) was observed along the DEJ in the presence of CGS27023A
Possible Involvement of Basement Membrane Damage by Matrix Metalloproteinases and Serine Proteinases
Involvement of Plasminogen Activator/ Plasmin in Damage to Epidermal BM, Degradation of Laminin 332, and Reduced Activities of Laminin 332 in Keratinocyte Adhesion and Type VII Collagen Binding UVB exposure increases the synthesis of urokinase-type plasminogen activator (uPA) [23, 42, 43], as well as matrix metalloproteinases [40, 44]. Both uPA activity and uPA are present in the conditioned medium of skin equivalents, and the addition of plasminogen enhances the degradation of BM components and impairs the assembly of BM structure at the DEJ even in the presence of MMP inhibitor (> Fig. 12.4) [42]. Aprotinin, a plasmin inhibitor, restores the assembly of BM structure at the DEJ damaged by the addition of plasminogen (> Fig. 12.4). Since lamina densa structure is detached at the anchoring filament from basal keratinocytes in sun-exposed skin (> Fig. 12.1), laminin 332, a major component of anchoring filament, is a candidate target of plasmin. As expected, plasmin degrades the a3 and b3 chains of laminin 332, although g2 is unaffected (> Fig. 12.5). Plasmin cleaves both the amino and carboxy terminals of a3 chain, and these cleavages reduce the keratinocyte adhesion activity of laminin 332 (> Fig. 12.6). Similarly, the removal of an amino terminal fragment (domain VI) of the b3 chain by plasmin (> Fig. 12.5) results in a reduction of its affinity for type VII collagen (> Fig. 12.6). Therefore,
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degradation of laminin 332 by plasmin may induce BM damage such as the detachment of lamina densa from basal keratinocytes.
Enhanced BM Assembly in the Presence of Laminin 332 and in Response to Increased Synthesis of BM Components in a Skin-Equivalent Model Damaged BM must be repaired, since BM at the DEJ plays important roles in maintaining a healthy epidermis and dermis. In order to find substances that stimulate the repair of damaged BM, a skin-equivalent model was selected for screening purposes, which is suitable for investigating the assembly of BM through cooperation between keratinocytes and fibroblasts. Purified laminin 332, a glycoprotein (MW: 410 kDa) composed of 165 kDa (a3), 140 kDa (b3), and 105 kDa (g2) chains, enhances the formation of hemidesmosome-like structures and BM at the DEJ in the skin equivalent [29]. Keratinocytes synthesize BM components, except nidogen [45]. Fibroblasts also produce BM components, other than laminin 332. Recently, it was found that increasing the production of BM components such as laminin 332, collagen IV, and collagen VII in keratinocytes and/or fibroblasts with/without inhibitors of gelatinases and/or serine proteinases is also effective to enhance the repair or assembly of BM at the DEJ.
. Figure 12.4 Ultrastructural analyses of BM structures at the DEJ of SE. Fourteen days after plating keratinocytes, the SEs were processed for the analysis of BM structures at the DEJ using electron microscopy. CGS27023A (10 mM) enhances the formation of a linear and continuous lamina densa-like structure (indicated by arrows) at the DEJ just beneath the basal keratinocytes. The addition of plasminogen (0.06 mM) impaired the assembly of the BM structure at the DEJ. Aprotinin (1.5 mM), a serine proteinase inhibitor, restored the linear and continuous lamina densa-like structures (indicated by arrows) at the DEJ
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. Figure 12.5 Degradation of laminin 332 by plasmin and analyses of the cleavage sites of laminin 332. The processed form of laminin 332 was purified from a conditioned medium of human keratinocytes and incubated with plasmin for 24 h at 37 C. SDS-PAGE on 5% acrylamide gel under reducing conditions and western blotting using polyclonal anti-laminin 332 antibodies were carried out. Lane 1, processed form of laminin 332; lane 2, plasmin-treated laminin 332. From the N-terminal amino acid sequences of the separated 145, 140, 110, and 105 kDa bands, cleavage sites in a3, b3, and g2 are indicated by arrows on schematic domain structures of laminin 332. Predicted cleavage sites of a3 chain are shown in G-3. Since the N-terminal sequence of a3 chain is DSSPA, which is the same as that of the 150 kDa fragment of the a3 chain, plasmin cleaved a 5 or 10 kDa fragment from the LG3 domain of the 150 kDa a3 chain with the putative cell adhesion region, LRD, is present
. Figure 12.6 Dysfunction of plasmin-treated laminin 332. Laminin 332 degraded by plasmin showed lower keratinocyte adhesive activity than control laminin 332. Purified plasmin-treated laminin 332 (open circle) and control laminin 332 (closed circle) were coated on 96-well plates and keratinocytes were added. The attached cells were quantitatively measured with the AlamarBlue assay. Laminin 332 degraded by plasmin (open circle) showed reduced binding to type VII collagen as compared with control laminin 332 (closed circle). Purified type VII collagen was coated on 96-well plates and purified plasmin-treated laminin 332 (open circle) or control laminin 332 (closed circle) was added. After washing, bound laminin 332 was detected using polyclonal anti-laminin 332 antibodies
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. Figure 12.7 Schematic representation of the involvement of BM damage in the process of photoaging. The disruption and reduplication of BM at the DEJ in sun-exposed skin may be induced by increased levels of BM-damaging enzymes, such as plasmin and MMPs, through the degradation of BM components: laminin 332, and type IV and VII collagens. The impairment of the BM structure may be associated with functional changes of the epidermal and dermal cells and consequently, facilitating aging processes by damaging dermal extracellular matrices and inducing keratinocyte abnormality
Conclusion The disruption and reduplication of BM at the DEJ in sun-exposed skin may be associated with increases of BM-damaging enzymes, such as plasmin and MMPs, which degrade BM components (laminin 332, and type IV and VII collagens). The impairment of BM structure may be associated with functional changes of epidermal cells and dermal cells and consequently may facilitate aging processes by damaging dermal extracellular matrices and inducing keratinocyte abnormality, as summarized in > Fig. 12.7. Laminin 332 is able to enhance BM assembly [29]. Some cosmetic ingredients also promote BM repair by increasing synthesis of BM components such as laminin 332, and type IV and VII collagens in the epidermis and/or the dermis. Therefore, the BM represents a good target for skin-care cosmetics; components that enhance BM repair may improve epidermal– dermal communication and skin homeostasis, thereby strengthening defenses against ‘‘skin aging.’’
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29. Tsunenaga M, Adachi E, Amano S, et al. Laminin 5 can promote assembly of the lamina densa in the skin equivalent model. Matrix Biol. 1998;17:603–613. 30. Tsunenaga M, Kohno Y, Horii I, et al. Growth and differentiation properties of normal and transformed human keratinocytes in organotypic culture. Jpn J Cancer Res. 1994;85:238–244. 31. Nishiyama T, Amano S, Tsunenaga M, et al. The importance of laminin 5 in the dermal-epidermal basement membrane. J Dermatol Sci. 2000;24:S51–59. 32. Feldman D, Bryce GF, Shapiro SS. Mitochondrial inclusions in keratinocytes of hairless mouse skin exposed to UVB radiation. J Cutan Pathol. 1990;17:96–100. 33. Sarret Y, Woodley DT, Goldberg GS, et al. Constitutive synthesis of a 92-kDa keratinocyte-derived type IV collagenase is enhanced by type I collagen and decreased by type IV collagen matrices. J Invest Dermatol. 1992;99:836–841. 34. Sudbeck BD, Parks WC, Welgus HG, et al. Collagen-stimulated induction of keratinocyte collagenase is mediated via tyrosine kinase and protein kinase C activities. J Biol Chem. 1994;269: 30022–30029. 35. Amano S, Ogura Y, Akutsu N, et al. Protective effect of matrix metalloproteinase inhibitors against epidermal basement membrane damage: skin equivalents partially mimic photoageing process. Br J Dermatol. 2005;153(2):37–46. 36. Herrmann G, Wlaschek M, Lange TS, et al. UVA irradiation stimulates the synthesis of various matrix-metalloproteinases (MMPs) in cultured human fibroblasts. Exp Dermatol. 1993;2:92–97. 37. Kawaguchi Y, Tanaka H, Okada T, et al. The effects of ultraviolet A and reactive oxygen species on the mRNA expression of 72-kDa type IV collagenase and its tissue inhibitor in cultured human dermal fibroblasts. Arch Dermatol Res. 1996;288:39–44. 38. Brenneisen P, Wenk J, Klotz LO, et al. Central role of Ferrous/Ferric iron in the ultraviolet B irradiation-mediated signaling pathway leading to increased interstitial collagenase (matrix-degrading metalloprotease (MMP)-1) and stromelysin-1 (MMP-3) mRNA levels in cultured human dermal fibroblasts. J Biol Chem. 1998; 273:5279–5287. 39. Koivukangas V, Kallioinen M, Autio-Harmainen H, et al. UV irradiation induces the expression of gelatinases in human skin in vivo. Acta Dermatol Venereol. 1994;74:279–282. 40. Fisher GJ, Datta SC, Talwar HS, et al. Molecular basis of sun-induced premature skin ageing and retinoid antagonism. Nature. 1996;379:335–339. 41. Inomata S, Matsunaga Y, Amano S, et al. Possible involvement of gelatinases in basement membrane damage and wrinkle formation in chronically ultraviolet B-exposed hairless mouse. J Invest Dermatol. 2003;120:128–134. 42. Ogura Y, Matsunaga Y, Nishiyama T, et al. Plasmin induces degradation and dysfunction of laminin 332 (laminin 5) and impaired assembly of basement membrane at the dermal-epidermal junction. Br J Dermatol. 2008;159:49–60. 43. Miralles F, Parra M, Caelles C, et al. UV irradiation induces the murine urokinase-type plasminogen activator gene via the c-Jun N-terminal kinase signaling pathway: requirement of an AP1 enhancer element. Mol Cell Biol. 1998;18:4537–4547. 44. Scharffetter K, Wlaschek M, Hogg A, et al. UVA irradiation induces collagenase in human dermal fibroblasts in vitro and in vivo. Arch Dermatol Res. 1991;283:506–511. 45. Fleischmajer R, Schechter A, Bruns M, et al. Skin fibroblasts are the only source of nidogen during early basal lamina formation in vitro. J Invest Dermatol. 1995;105:597–601.
11 Proteoglycans in Skin Aging Franc¸ois-Xavier Maquart . Ste´phane Bre´zillon . Yanusz Wegrowski
Introduction Proteoglycans are ubiquitous macromolecules of extracellular matrices, cell surfaces, and some intracellular granules [1]. They are composed of a glycoprotein core to which one or several sulfated glycosaminoglycan (GAG) chains are attached by covalent linkage. Four different classes of sulfated glycosaminoglycans (GAGs) exist in vertebrates: chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and heparan sulfate/heparin (HS). Hyaluronic acid is not esterified with sulfate and not linked to a protein core. The properties, structure, and functions of glycosaminoglycan are discussed in another chapter. One protein core may bear one type or several types of glycosaminoglycans of the same or different classes. For example, syndecan-1 may contain heparan sulfate only or heparan sulfate and chondroitin sulfate glycosaminoglycan chains. Depending on cell context, the same proteoglycan may be substituted with different GAG chains. For example, serglycin of mast cells is substituted with heparin and serglycin of macrophages is substituted with chondroitin sulfate. A third feature of these macromolecules is the possibility of absence of the glycosaminoglycan chain in the same proteoglycan, depending on the organ. Lumican in cornea is a keratan sulfate proteoglycan, whereas in skin it is a glycoprotein without any glycosaminoglycan chain. Finally, the presence of GAG may be time dependent in the so-called part-time proteoglycans. Often, it is done by the synthesis of splicing variants lacking the GAG attachment sequence, as in the versican V3 variant. The GAG chains of galactosaminoglycans (CS/DS) and HS are attached to a serine residue of the core protein via a short common tetrasaccharide, by a beta 1–4 bond in the case of CS/DS and by alpha 1–4 bond in the case of HS. This region is composed of a tetrasaccharide GlcA (b1-3)Gal(b1-3)Gal(b1-4)Xylb1-O-Ser, where GlcA is glucuronic acid, Gal is galactose, and Xyl is xylose. Keratan sulfate chains of aggrecan (KS type II) are also attached to a serine residue via O-glycosidic linkage, but the composition of this linkage is similar to those of the O-substituted oligosaccharides of glycoproteins. Keratan sulfate chains of lumican or keratocan in cornea (KS I)
possess a mannose-rich region attached by N-glycosidic linkage to an asparagine, similar to the N-substituted oligosaccharides of glycoproteins. Although the presence of keratan sulfate in skin has been reported [2], no detailed structure of the linkage region of these molecules is known. Several glyco and sulfotransferases are involved in the assembly of the GAGs on the protein core. The xylosyltransferases are the enzymes, which start the GAG synthesis. Although there is no consensus sequence for a given exported protein to be substituted with GAG chain, the xylosyl-transferases recognize Ser-Gly sequences in the vicinity of acid-amino acids [3]. It seems that the presence of hydrophobic clusters and the conformation of the protein also contribute to the activity of N-acetyl-glucosamine transferase I and N-acetyl-galactosamine transferase I, two enzymes which initiate the synthesis of HS or CS/DS, respectively. At present, over 50 proteoglycan genes are known. The mRNAs of most of them are expressed in skin cells (> Table 11.1) [4]. However, only some of the protein products have been confirmed to reside in the skin. The extracellular proteoglycans are regrouped in different groups or classes. First and most abundant class of extracellular matrix proteoglycans of the dermis belong to the small, leucine-rich proteoglycan (SLRP) family [1]. Although there is no comparative quantification of different proteoglycans of human dermis, decorin seems to be the most aboundant representative in the skin, associated with collagen fibers. The same localization concerns lumican, which, in the skin, carries no GAG chain. Biglycan is present mostly in the skin pericellular matrix [5]. Fibromodulin was recently reported to be present in the epidermis [6]. Second group contains large, aggregating proteoglycans that are able to bind hyaluronic acid, usually called hyalectans (or lecticans). These proteoglycans are able to form very high molecular mass complexes, composed of numerous proteoglycan molecules settled down on a hyaluronan molecule [1]. From this group, only versican was documented in human dermis and epidermis [7]. Basement membrane of the dermo–epidermal junction contains a major heparan sulfate proteoglycan: perlecan
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_11, # Springer-Verlag Berlin Heidelberg 2010
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. Table 11.1 The proteoglycans mRNA expressed by keratinocytes (K) or skin fibroblasts (F) screened by cDNA array obtained from GEO profiles of NCBI [4] (A) Extracellular proteoglycans
Gene
NCBI Ref.Seq
mRNA expression
GAG chain
SLRP class Decorin
DCN
NM_001920a
K/F
DS/CS
Biglycan
BGN
NM_001711
K/F
DS/CS
Asporin
ASPN
NM_017680
K/F
CS
Extracellular matrix protein 2
ECM2
NM_001393
K
Fibromodulin
FMOD
NM_002023
K
Lumican
LUM
NM_002345
F
KS
Pro/Arg-rich end LRR protein
PRELP
NM_002725a
K
CS
Keratocan
KERA
NM_007035
K/F
KS
Osteomodulin
OMD
NM_005014
K
CS
Epiphycan
EPYC
NM_004950
K/F
CS
a
KS
Osteoglycin
OGN
NM_033014
F
CS
Chondroadherin
CHAD
NM_001267
K
CS
Nyctalopin
NYX
NM_022567
K/F
VCAN
NM_004385a
F
a
Hyalectans Versican
CS/(DS)
Aggrecan
ACAN
NM_013227
K
CS + KS
Neurocan
NCAN
NM_004386
K
CS
Brevican
BCAN
NM_021948a
K/F
CS
Perlecan (heparan sulfate proteoglycan 2)
HSPG2
NM_005529
K/F
HS/(CS)
Agrin
AGRN
NM_198576
K/F
HS
Bamacan (structural maintenance of chromosome 3)
SMC3
NM_005445
K/F
CS
Leprecan (PRP, prolyl-3-hydroxylase)b
LEPRE1
NM_022356a
K/F
CS
Type IX
COL9A2
NM_001852
K
CS
Type XII
COL12A1
NM_004370
F
CS
Basement membrane PGs
b
Collagens
Type XIV
COL14A1
NM_021110
F
CS
Type XVIIIb
COL18A1
NM_030582a
K/F
HS
Testican 1
SPOCK1
NM_004598
K/F
CS
Testican 2
SPOCK2
NM_014767a
K
CS
Adlican
MXRA5
NM_015419
K/F
?
Colony simulating factor-1 (CSF-1, M-CSF)
CSF-1
NM_000757a
K/F
CS
Testicans
Miscellaneous
(B) Cell membrane proteoglycans
Gene
NCBI Ref.Seq
mRNA expression
GAG chain
Syndecans Syndecan-1
SDC1a
NM_001006946
K/F
HS/(CS)
Syndecan-2
SDC2
NM_002998
F
HS
Proteoglycans in Skin Aging
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. Table 11.1 (Continued) (B) Cell membrane proteoglycans
Gene
NCBI Ref.Seq
mRNA expression
GAG chain
Syndecan-3
SDC3
NM_014654
K/F
HS
Syndecan-4
SDC4
NM_002999
K/F
HS
Glypican-1
GPC1
NM_002081
K/F
HS
Glypican-3
GPC3
NM_004484
K
HS
Glypican-4
GPC4
NM_001448
K
HS
Glypican-5
GPC5
NM_004466
K
HS
Glypican-6
GPC6
NM_005708
F
HS
CD44
NM_000610a
K
CS/HS
Neuroglycan C chondroitin sulfate proteoglycan 5
CSPG5
NM_006574
K
CS
Phosphacan
PTPRZ1
NM_002851
K
CS
ENG,
NM_000118a
K/F
CS
Glypicans
Hyalectans CD44 (lymphocyte homing receptor) Neural PGs
Endothelial PGs Endoglin (CD105)
a
Endothelial cell-specific molecule 1 (endocan)
ESM1
NM_007036
K
DS
Thrombomodulin (CD 141)
THBD
NM_000361
K
HS
NG2 (melanoma-associated antigen, MAA), chondroitin CSPG4 sulfate proteoglycan 4
NM_001897
K/F
CS
Betaglycan
NM_003243
K
CS
Miscellaneous
(C) Granules proteoglycan Serglycin
TGFBR3 Gene SRGN
NCBI Ref.Seq
mRNA expression
GAG chain
NM_002727
K
Heparin/ CS
a
Different splicing variants or mRNAs Basement membrane-associated proteoglycans
b
(see below). Some collagens (types IX, XII, XIV, and XVIII) also contain a covalently linked GAG chain. Apart from type IX collagen, they are all expressed in collagen-rich dermis [8]. Testicans form another family of extracellular proteoglycans. Four testican genes have been cloned, but only testican-1 and testican-2 mRNAs are expressed in the skin, by keratinocytes (> Table 11.1). Among miscellaneous proteoglycans, the mRNA of adlican was reported to be expressed in the fibroblasts of very old subjects, but nothing is known about the protein. Another proteoglycan, CSF-1, is expressed by resident skin cells of the immune system, e.g., Langerhans cells [9]. Cell membrane proteoglycans, which are characterized by a very rapid turnover, include two main families of heparan sulfate macromolecules. The family of syndecans (four genes cloned) comprises integral type I membrane proteins, with a transmembrane domain and a
short cytoplasmic tail (> Table 11.1). Syndecan-1 is expressed in epidermis including hair follicles [10]. The family of glypicans (six genes cloned) has glycosylphosphatidylinositol-anchored molecules with a glycanic chain attached at the vicinity of plasma membrane [11]. Only the expression of neural glypican-2 was not detected in the skin (> Table 11.1). Syndecans and glypicans are easily shedded by the action of metalloproteinases and phospholipases, respectively, and are often found in pericellular space. CD44 and one of its splicing epidermal variants, epican, is an integral plasma membrane proteoglycan, which binds hyaluronic acid [12]. Keratinocytes also express the mRNAs of two brain proteoglycans, neuroglycan-C and phosphacan (> Table 11.1). One of the splicing variants of phosphacan possesses protein tyrosine phosphatase activity. Some proteoglycans characteristic for endothelial cells are also
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expressed by keratinocytes. The proangiogenic transforming growth factor-beta (TGF-b) co-receptor, endoglin, is expressed in epidermis [13], as well as the thrombin receptor, thrombomodulin [14]. Another TGF-b co-receptor, betaglycan, also known as TGF-beta receptor type III, is also expressed in epidermis at the mRNA level, but the presence of the protein is not documented. Characteristic for embryonic development, the melanoma-associated antigen/NG2 is an integral membrane, chondroitin sulfate containing high molecular mass proteoglycan overexpressed in aggressive melanoma lesions [15]. NG2 was proposed to be a marker of melanoma invasion in the lymphatic glands. This proteoglycan is also a characteristic for stem cell population of hair follicles root sheet [16]. Skin is a niche of mast cells, which contain heparin in their secretory granules. Heparin is linked to the proteoglycan serglycin core protein. This is also expressed by macrophages and Langerhans cells but, in this case, it is substituted with chondroitin sulfate chains [1]. The knowledge about the roles of proteoglycans in physiologic and pathologic conditions started when the first demonstration of a dermatan sulfate–protein complex in the skin was done [17]. The cloning era, followed by the creation of knockout mice, permitted to this knowledge to expand rapidly. Numerous excellent reviews, either general or devoted to particular class/families of proteoglycans give up-to-date state of the art in this area (cited in [1]). In general, skin proteoglycans retain all the properties and functions of particular glycosaminoglycans, contributing to tissue hydratation, resistance and resilience, molecular filtration, cell behavior, and cell– cell or cell–matrix interaction. Additionally, by their polyanionic properties, they contribute to cation exchange and to the retention of all positively charged proteins. As a consequence, they constitute the biological reservoir of many cytokines and growth factors. Such a diversity of functions supposes that any changes of their expression or structure during aging may perturb the tissue homeostasis in important manner. The clinically relevant morphological changes of the skin during aging can be summarized by the term ‘‘senile atrophy.’’ Main changes are a diminished thickness of epidermis with a reduced mitosis rate of epidermal basal cells and a decreased number of fibroblasts and capillaries in the dermis. The mesenchymal changes in the dermis have been morphologically described by the term ‘‘senile elastosis’’ or ‘‘elastoid collagen degeneration,’’ corresponding to a progressive collagen denaturation with aging. Analysis of the glycosaminoglycan content of the senile skin showed a minimal increase of the total content of hexosamines and
uronic acids with a significant increase of DS and KS, a decrease of hyaluronic acid and, also partly, a decrease of chondroitin-4-sulfate and chondroitin-6-sulfate. The neosynthesis of sulfated glycosaminoglycans is only slightly increased in aged skin, whereas the activities of the enzymes specific for the glycosaminoglycan catabolism (beta-glucuronidase, beta-N-acetyl-glucosaminidase) are significantly decreased [18].
Proteoglycans of the Dermis Extracellular Matrix Small Leucine-Rich Proteoglycans Small leucine-rich proteoglycans (SLRPs) are a family of proteoglycans present in a large number of tissues. They are characterized by their relatively low molecular weight and by the presence of similar structural motifs, the leucine-rich repeats (LRR) [19]. They are presently classified into five distinct families [20], according to their structural features and chromosomal organization (> Table 11.2). Dermis contains several SLRPs, particularly decorin and biglycan [5], lumican [21], and keratocan [22]. SLRPs . Table 11.2 Classification of human Small Leucine-rich proteoglycans (SLRPs) Schaefer L, Iozzo RV. [20] SLRP class I
Name Biglycan Decorin Asporin ECM2
II
Fibromodulin Lumican PRELP Keratocan Osteoadherin
III
Epiphycan Opticin Osteoglycin
IV
Chondroadherin Nyctalopsin Tsukushi
V
Podocan Podocan-like protein 1
Proteoglycans in Skin Aging
play important roles in the regulation of cell activity and in the organization and functional properties of skin connective tissue [23]. Several lines of mice deficient in SLRPs have been generated, particularly for the most prominent and widely expressed SLRPs: decorin, biglycan, fibromodulin, and lumican. All these SLRP deficiencies result in the formation of abnormal collagen fibrils and induce a wide array of diseases in the deficient mices (for review, see [24]). The implication of SLRPs modifications in the alterations of skin function, which may occur in skin aging has been suggested by several studies devoted to decorin and, more recently lumican.
Decorin Decorin was the most studied among the SLRP family. It is composed of a 40 kDa protein core, bearing a unique DS/CS chain. It is the most abundant SLRP in adult human dermis, where it is observed in association with collagen fibers. It is synthesized and secreted by dermal fibroblasts and might represent 30–40% of total proteoglycans of the skin [25]. Previous data from Bernstein et al. [26] reported that decorin was greatly decreased within photoaged skin, as appreciated by immunohistochemical staining and confocal laser scanning microscopy. In addition, Northern blot analysis showed that decorin steady-state mRNA levels measured in fibroblasts derived from photoaged skin was decreased by 46% in cultures derived from photo-damaged sites. Since collagen degradation was strongly increased in such areas, and collagen amounts strongly decreased, it is possible, however, that the amount of decorin necessary to bind available collagen fibers may decrease in photoaged skin as collagen degradation takes place. The decorin alterations in skin chronological aging seem very different from that observed in photoaging. As soon as 1997, Passi et al. [27] studied the modifications of proteoglycans secreted into their growth medium by young and senescent fibroblasts. In this study, senescence was induced in human skin fibroblasts by serial passages in culture, and their proteoglycan synthesis was studied by incorporation of radiolabeled precursors. The authors observed that, whereas the biosynthesis of total proteoglycan fraction was not altered with replicative senescence, the relative proportions of the different proteoglycan populations secreted into the growth medium changed. Particularly, the relative content of small CS/DS-proteoglycans that consisted mainly of decorin, as shown by immunological identification, was increased by 50% in senescent (28–31 passages) versus young (4–5 passages)
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fibroblasts. Interestingly, the authors suggested that the increased secretion of decorin into the growth medium by the late passage fibroblasts might contribute to their decline of proliferative capacity, a characteristic common to every aging cells. In a study of human skin samples from fetal skin, mature skin, and senescent skin, Carrino et al. [28] showed an increase in the proportion of decorin with a concomitant decrease of the large chondroitin sulfate proteoglycan, versican, in senescent skin. In addition, the authors reported that decorin from postnatal skin is smaller in size than the decorin of mature skin, probably due to shorter glycosaminoglycan chains. More importantly, the authors reported the presence in aged skin of a shortened (Mr about 45 kDa) form of decorin, with a core protein of about 27 kDa only. Analysis of this molecule, named ‘‘decorunt’’ by the authors, indicated that it was a catabolic fragment of decorin, representing the amino-terminal 43% of the mature decorin molecule [29]. Decorunt contains the first four leucine-rich repeats and three amino acids of the fifth leucine-rich repeat. Since decorunt lacks almost half of the collagen-binding domain of decorin, it was expected to have impaired capacity to stabilize collagen fibers. Moreover, since the sixth leucine-rich repeat, the most important for the interaction between decorin and the epidermal growth factor receptor, is lacking, decorunt should be unable to bind to this receptor, which may induce a defect of cell stimulation in aged skin. In addition, the absence of the transforming growth factor-b binding site in decorunt might impair the sequestering of this growth factor in the extracellular matrix. In an immunohistochemical study of rat dermis, Ito et al. [30] reported that decorin was only faintly visible in young (22 days old) rats, whereas it was abundant on the collagenous network of the dermis of old (24–30 months old) rats. Since decorin was shown to have a growth inhibitory effect, the authors suggested that decorin excess might be one of the cell growth inhibitory factors implicated in skin senescence. Analysis of rat skin performed by Nomura et al. [31] confirmed that the amount of decorin in rat skin increased with rat’s age (postpartus 0.5 days until 90 days of age). Interestingly, the authors noted that the molecular size of decorin decreased during aging, from about 111 kDa in 18.5 days embryo to about 70 kDa at postpartus 90 days. This decrease of size was not due to a decreased size of the core protein but to a decreased length of the glycosaminoglycan chain. The mean length of the chain was 78.58 13.94 nm in the skin of the 18.5 days embryos versus 54.05 4.79 nm in the 90 days postpartus
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rat skin. Reducing the length of the decorin glycosaminoglycans was supposed to reduce the distance between the collagen fibers [32]. In a clinical study performed on full thickness punch biopsies obtained from five young (25–35 years) and five older (61–68 years) volunteers, Lockner et al. [33] reported that decorin mRNA in skin biopsies from older volunteers was approximately 100% higher that in younger volunteers. In contrast, type I and type III collagens mRNAs were decreased. The authors suggested that the resulting decreased collagen to decorin ratio in older skin might be responsible for decreased collagen bundle diameters in aged human skin, which might affect their tensile strength [34, 35].
Lumican Lumican was first identified as a major proteoglycan of the cornea [36]. It is present in this tissue under the form of a keratan-sulfate (KS)-containing proteoglycan, with a 38 kDa core protein. In adult human skin, however, it is present under the form of a glycoprotein, devoid of glycosaminoglycan chain [37]. As decorin, lumican seems to play an important role in the preservation of skin functional properties. For instance, lumican-null mice showed abnormal collagen fibril assembly, with large and abnormally shaped collagen fibrils and an extremely loose and fragile skin [38]. In a recent study, Vuillermoz et al. [39] studied the expression of SLRPs by early passage cultured fibroblasts, obtained from 36 normal donors of 1 month to 83 years old. By Northern blot analysis, the authors showed a significant negative correlation of lumican mRNA expression with donor’s age. By contrast, no correlation with age was found for either decorin or biglycan mRNAs. Immunohistochemistry associated with image analysis quantitation showed that lumican core protein was preferentially located in the superficial, papillary layer of the dermis and that it was strongly decreased ( 81% and 85% respectively, p < 0.01) in aged skin (donors over 50) compared to young (0–15 years old) and adult (16–50 years old) skin. The strong decrease of lumican expression in aged skin, associated with the increase of decorin, induced important modification of the lumican to decorin ratio. Due to the particular importance of these two SLRPs in dermal organization and properties, the authors suggested that these alterations might be involved in the functional defects, which characterize aged skin.
Versican and Other Proteoglycans of the Dermis Extracellular Matrix Although skin fibroblasts express mRNAs for different proteoglycans (> Table 11.1) only type XII and XIV collagens and versican were studied in the dermis. Versican or chondroitin sulfate-glycoprotein 2 (CS-PG2), or PG-M, has a tridomain structure. The amino-terminal end, designated as G1, binds to hyaluronan. The carboxy-terminal domain, G3, possesses a lectin domain adjacent to two epidermal growth factor domains, and a complement regulatory domain. The central domain (G2) is encoded by two exons that specify CS attachment regions. RNA splicing of these two exons results in four different forms of versican, called V0–V3 (molecular weight 370, 262, 180, and 72 kDa, respectively) with different numbers of GAG attachment domains [40–42]. The distribution of the V0–V3 forms in adult varies with tissues and organs. Versican is a widespread ECM components of rodent and human skin and has also been characterized in the kidney in the lamina propria of blood vessels [43]. Versican expression was reported to decrease in aging skin [26, 28]. However, in photoaged skin, an accumulation of versican in elastotic material was reported [44]. After acute UV irradiation, there was a rapid upregulation of versican mRNA, which persisted up to 72 h after exposure. The accumulation of versican was accompanied by the formation of a truncated molecule, which lacked the hyaluronan-binding region of this proteoglycan [45]. In chronic sun exposure, this accumulation of versican led to the decrease of its synthesis, accelerating the loss of the molecule. As versican plays an important role in cell–hyaluronan interaction, the loss of this proteoglycan may substantially contribute to the loss of skin elasticity and resilience, especially in photoaged skin.
Cell Surface Proteoglycans in Skin Aging Cell surface proteoglycans may be divided into two main families: the syndecans and the glypicans. The main structural difference between both families is that syndecans have a transmembrane core protein, whereas, for glypicans, the core protein is linked to the cell surface by a glycosyl-phosphatidyl-inositol (GPI) anchor. Both families belong to the HS proteoglycans group even if syndecans may carry CS glycosaminoglycan chains in addition to HS.
Proteoglycans in Skin Aging
Syndecans In vertebrates, the syndecan family is composed of four members: syndecan-1, -2, -3, and -4 (for recent review, see [46]). Each core protein is composed of an extracellular domain bearing three to five chains of HS and CS, a single-span transmembrane domain and a short cytoplasmic domain. The molecular weight of the four syndecan core proteins are respectively 33, 23, 43, and 22 kDa for syndecans 1, 2, 3, and 4. The extracellular domains of syndecans are able to interact with a number of extracellular proteins such as fibronectin and growth factors, mainly fibroblast growth factors (FGFs) and vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-b), and platelet-derived growth factor (PDGF). These interactions essentially occur through the heparan-sulfate chains of syndecans. On the other hand, the intracellular domain is able to bind to other syndecan molecules, which permits their multimerization, and to a number of intracellular molecules, involved either in cytoskeleton formation or in intracellular signaling. Recent data indicates that syndecans are involved in complex signaling and cytoplasmic interactions, and could be present in membrane microdomains specialized in signal transduction [47]. Syndecan-1 is the most abundant syndecan on epithelial keratinocytes. It is strongly expressed in the suprabasal, differentiating epidermal cells, whereas it is expressed at low levels in the basal layer [48, 49]. Its expression is, however, markedly upregulated in all cell layers of the epidermis during wound healing [50]. Recent data demonstrated that overexpression of syndecan-1 in transgenic mouse epidermis induces epidermal proliferation, as evidenced by increased number of suprabasal cell layers, elevated proliferating cell nuclear antigen (PCNA) expression in both basal and suprabasal cell layers. The expression of terminal differentiation markers, keratin 10 and involucrin, was not disrupted in the epidermis of transgenic animals, showing that epidermal differentiation was not altered [51]. On the other hand, the loss of syndecan-1 in syndecan-1-null mice was shown to induce a decreased migration rate, linked to an increased adhesion of syndecan-1-defective keratinocytes [52]. Taken together, these results suggest that syndecan-1 is an important modulator of epidermal cell proliferation, migration, and adhesion and may control the behavior of epithelial cells. It may be suggested that the decreased proliferation potential of keratinocytes in aging epidermis might be linked to a defect in syndecan-1 expression.
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Syndecan-4 is another important component of the skin. In normal skin, it is detectable in the epidermis but not in the dermis. It is, however, strongly expressed by fibroblasts and endothelial cells in wounded skin [53]. Mice lacking syndecan-4 are characterized by delayed wound repair and impaired angiogenesis [54], which demonstrate that this proteoglycan is essential for skin repair. This finding may be important for understanding the physiopathology of skin aging. Few works have been devoted to the variations of syndecan expression in aging skin. Preliminary data from the authors’ laboratory indicated, however, that syndecan expression may be altered during aging. Reverse transcription polymerase chain reaction (RT-PCR) analysis of syndecan-1 mRNA indicated that keratinocytes may express syndecans 1, 2, and 4. Syndecan-1 expression was decreased in keratinocytes from donors over 50 years old [55]. That decrease was confirmed by immunocytochemical analysis of syndecan-1 in the epidermis of donors over 50 compared to donors between 16 and 50 years old [56]. Such a decrease in syndecan-1 might alter signalization processes in aged skin.
Glypicans Glypicans were identified about 15 years ago as phosphatidyl-inositol-anchored membrane heparansulfate proteoglycans [57]. In mammals, the glypican family is presently composed of six members, glypican-1 to glypican-6, with a core protein of 60–70 kDa [11]. The insertion sites for the heparan sulfate chains seem to be restricted to the last 50 amino acids in the C-terminus, placing the chains close to the cell membrane [58]. Glypicans are predominantly expressed during development [57]. Their main function is to regulate the signaling of Wnts, Hedgehogs, fibroblast growth factors, and bone morphogenetic proteins. Depending on the context, they may have either a stimulatory or inhibitory activity on signaling [11]. Part of glypicans may be shedded from the cell membrane into the extracellular matrix. This shedding is dependent on an extracellular lipase, Notum [59]. Little data is available about the presence of glypicans in the skin and their function. Preliminary data from the authors’ laboratory, however, indicated that human keratinocytes may express glypicans 1, 3, 4, 5, and 6 mRNAs [55]. However, no variation was observed on glypican expression during skin aging.
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Previous data from Litwack et al. [60] detected glypican-1 mRNA and protein expression in rat skin. Significant glypican-1 protein staining was detected in all layers of epidermis, except stratum corneum, and slight expression was found in the dermis. No study of glypican variation during aging was, however, performed by the authors.
Role of the Heparan-Sulfate Chains Since both syndecan and glypican are heparan-sulfate proteoglycans, these molecules may bind and modulate the activity of several matrix components, growth factors, proteinase inhibitors, cell–cell, and cell–matrix adhesion molecules [61]. Such an ability likely plays an important role in the regulation of skin cell functions, especially in the aging process. Further investigations have, however, to be performed to better precise the exact place of the molecules in skin physiology.
Proteoglycans of the Dermo–Epidermal Junction Components of the dermo–epidermal junction include collagen IV, collagen VII, laminin, entactin/nidogen, fibronectin, as well as subepidermal dermal markers (collagen I and fibrillin 1), and proteoglycans (mainly HS proteoglycans). Collagen type IV, HS proteoglycans, laminin, entactin/nidogen, and fibronectin are products of the epidermal cells of the skin [62].
Heparan-Sulfate Proteoglycans (HS-PGs) HS-PGs are common constituents of cell surfaces and of the ECM, including the basement membrane [63, 64]. The HS-PGs found in the ECM are perlecan, agrin, and collagen XVIII [65, 66]. HS-PGs are implicated in regulating the integrity of basement membranes, morphogenesis, angiogenesis, tumor metastasis, and tissue repair. These activities are attributed to the ability of HS-PGs to bind mitogenic and angiogenic growth factors [67], and to modulate their biological activities. Severe structural changes, including deterioration of the mechanical properties of the dermis, occur during skin aging. Skin HS-PGs, which regulate cell proliferation and proteolysis, as well as matrix adhesion and assembly decrease during aging and thus, may be implicated in the functional alterations linked to the aging process. They
may represent important targets in dermo-cosmetology for fighting skin aging. For instance, Pineau et al. [68] demonstrated the potential interest of a new C-xylopyranoside derivative (C-beta-D-xylopyranoside2-hydroxy-propane, simplified as C-xyloside) to improve HS-PG production in human skin. In an organotypic model of corticosteroid atrophied human skin, characterized by a decrease of PGs expression, treatment with C-xyloside improved expression of HS-PGs. Improvement of the dermo–epidermal junction in human reconstructed skin treated by C-xyloside was also reported [69]. Basement membranes contain several proteoglycans, and those bearing heparan-sulfate chains such as perlecan and agrin, usually predominate.
Perlecan Perlecan is a large proteoglycan, with a core protein of 396 kDa, divided into five domains. It is predominantly substituted with HS chains, but on some occasions it may be substituted with CS, DS, hybrid HS/CS, or CS/DS. It may be also secreted as a glycosaminoglycan-free glycoprotein [70, 71]. Perlecan is present in virtually all basement membranes [72, 73]. It interacts with basement membrane components such as laminin-1 and collagen IV, but also with cell adhesion molecules such as b1-integrin [74]. Inactivation of the perlecan gene in mice results in embryonic lethality and prenatal death. Similarly, the functional null mutation in the perlecan gene is characterized by perinatal death in humans [75]. Perlecan was shown to regulate both the survival and terminal steps of differentiation of keratinocytes: Sher et al. [76] demonstrated that perlecan regulates these processes via controlling the bioavailability of perlecan-binding soluble factors (particularly keratinocyte growth factor or FGF-7), involved in epidermal morphogenesis. Consequently, it may be suggested that perlecan is important for the preservation of skin epithelium. Pineau et al. [68] reported a decreased expression of perlecan during skin aging. This decrease might be involved in the alterations of basement membrane observed in aged skin.
Agrin Agrin is a ubiquitously expressed proteoglycan, with a 200 kDa core protein [77]. It contains not only HS chains,
Proteoglycans in Skin Aging
but also CS chains. The synaptic basal lamina, a component of ECM in the synaptic cleft at the neuromuscular junction, is rich in agrin. However, many other structures, which have basal laminae, including skin, also contain agrin. Available information indicates, however, that no data on agrin expression in skin aging has been published.
Collagen XVIII Type XVIII collagen is an ubiquitous basement membrane zone component, occurring prominently at vascular and epithelial basement membranes including dermo– epidermal junction [78]. While it contains collagen domains, it is also a member of the proteoglycan family since it carries a heparan-sulfate chain. Endostatin, a proteolytic fragment of type XVIII collagen, has been shown to inhibit angiogenesis, tumor growth, and endothelial cell proliferation and migration. Overexpression of endostatin in the skin [79] induced a widening of the epidermal basement membrane zone, as observed by electron microscopy. Immunoelectron microscopy of the type XVIII collagen in mouse skin showed a polarized orientation of this molecule in the basement membrane, with the C-terminal endostatin region localized in the lamina densa. In transgenic mice overexpressing endostatin, type XVIII collagen was dispersed in the skin, suggesting that the transgene-derived endostatin might displace the full-length collagen XVIII. This may impair the anchoring of the lamina densa to the dermis and thereby lead to loosening of the basement membrane zone, resembling the previously observed situation in collagen XVIII-null mice. It was reported that desorganization of the basement membrane zone was one of the features of aged skin [80].
Chondroitin-Sulfate Proteoglycans Although less abundant than HS-PG, several CS-PGs have been described in the basement membrane.
Versican General characteristics of versican were presented in Section ‘‘Versican and Other Proteoglycans of the Dermis Extracellular Matrix.’’ While mainly present in the dermis, traces of versican have been found in some basement
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membranes. No data is available, however, about alterations of versican in the dermo–epidermal junction of aging skin.
Leprecan Leprecan has been described as a CS-PG with a 100-kDa core protein [81]. Immunostaining with polyclonal antibodies showed the localization of its protein core into the basement membrane of the vasculature. Rat-homolog of Gros1, a human growth suppressor gene, was identified to be the leucine–proline-enriched basement membraneassociated proteoglycan leprecan [82]. Leprecan was predicted to be a protein hydroxylase that might be involved in the generation of substrates for protein glycosylation [83]. It was suggested that regulated expression of leprecan coupled with its reported prolyl hydroxylase activity might play a role during basement membrane assembly in the kidney [84]. Available information indicates, however, that no data about alterations of leprecan expression during skin aging has been published.
Bamacan Bamacan is a CS-PG that abounds in basement membranes. Bamacan can occur in certain cell types as either a secreted proteoglycan involved in basement membrane assembly or as an intracellular protein contributing to the structure of chromosome 3 (SMC3) [85]. The entire bamacan core protein is characterized by an Mr of 138 kDa. A stabilizing role for bamacan in the basement membrane has been proposed [86]. Available information indicates, however, that no data on bamacan expression during skin aging has been published.
Conclusion It is clear from the data reported in this chapter that proteoglycans may play a major role in skin homeostasis, and in the functional and architectural properties of this tissue. While many data are available concerning some particular proteoglycans of the dermal or epidermal compartments, for instance, decorin in the dermis or syndecans in the epidermis, large areas remain, however, unknown at the present time. It is particularly the case of the alterations of proteoglycan expression, distribution,
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or structure, which may occur during the aging process and, more importantly, on their potential consequences on skin physiology. Since these alterations may affect not only the glycosaminoglycan chains, but also the protein core of the proteoglycans itself, it will be important to design new experimental models, especially in vivo, which will permit to better study their consequences on skin physiology and their implication in the defects linked to skin aging.
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59. Traister A, Shi W, Filmus J. Mammalian Notum induces the release of glypicans and other GPI-anchored proteins from the cell surface. Biochem J. 2008;410:503–511. 60. Litwack ED, Ivins JK, Kumbesar A. Expression of the heparan sulfate proteoglycan glypican-1 in the developing rodent. Dev Dyn. 1998;211:72–87. 61. Jackson RL, Busch SJ, Cardin AD. Glycosaminoglycans: molecular properties, protein interactions and role in physiological processes. Physiol Rev. 1991;71:481–539. 62. Tamiolakis D, Papadopoulos N, Anastasiadis P, et al. Expression of laminin, type IV collagen and fibronectin molecules is related to embryonal skin and epidermal appendage morphogenesis. Clin Exp Obstet Gynecol. 2001;28:179–182. 63. David G. Integral membrane heparan sulfate proteoglycans. FASEB J. 1993;7:1023–1030. 64. Erickson AC, Couchman JR. Still more complexity in mammalian basement membranes. J Histochem Cytochem. 2000;48:1291–1306. 65. Blackhall FH, Merry CL, Davies EJ, Jayson GC. Heparan sulfate proteoglycans and cancer. Br J Cancer. 2001;85:1094–1098. 66. Jiang X, Multhaupt H, Chan E, et al. Essential contribution of tumor-derived perlecan to epidermal tumor growth and angiogenesis. J Histochem Cytochem. 2004;52:1575–1590. 67. Iozzo RV. Heparan sulfate proteoglycans: intricate molecules with intriguing functions. J Clin Invest. 2001;108:165–177. 68. Pineau N, Bernerd F, Cavezza A, et al. A new C-xylopyranoside derivative induces skin expression of glycosaminoglycans and heparan sulphate proteoglycans. Eur J Dermatol. 2008;18:36–40. 69. Sok J, Pineau N, Dalko-Csiba M, et al. Improvement of the dermal epidermal junction in human reconstructed skin by a new c-xylopyranoside derivative. Eur J Dermatol. 2008;18:297–302. 70. Isemura M, Sato N, Yamaguchi Y, et al. Isolation and characterization of fibronectin-binding proteoglycan carrying both heparan sulfate and dermatan sulfate chains from human placenta. J Biol Chem. 1987;262:8926–8933. 71. Iozzo RV, Hassell JR. Identification of the precursor protein for the heparan sulfate proteoglycan of human colon carcinoma cells and its post-translational modifications. Arch Biochem Biophys. 1989;269: 239–249. 72. Iozzo RV, Cohen IR, Gra¨ssel S, Murdoch AD. The biology of perlecan: the multifaceted heparan sulphate proteoglycan of basement membranes and pericellular matrices. Biochem J. 1994;302:625–639. 73. Murdoch AD, Liu B, Schwarting R, et al. Widespread expression of perlecan proteoglycan in basement membranes and extracellular matrices of human tissues as detected by a novel monoclonal antibody against domain III and by in situ hybridization. J Histochem Cytochem. 1994;42:239–249. 74. Brown JC, Sasaki T, Go¨hring W, et al. The C-terminal domain V of perlecan promotes beta1 integrin-mediated cell adhesion, binds heparin, nidogen and fibulin-2 and can be modified by glycosaminoglycans. Eur J Biochem. 1997;250:39–46. 75. Arikawa-Hirasawa E, Yamada Y. Roles of perlecan in development and disease: studies in knockout mice and human disorders. Seikagaku 2001;73:1257–1261. 76. Sher I, Zisman-Rozen S, Eliahu L, et al. Targeting perlecan in human keratinocytes reveals novel roles for perlecan in epidermal formation. J Biol Chem. 2006;281:5178–5187. 77. Godfrey EW, Dietz ME, Morstad AL, et al. Acetylcholine receptoraggregating proteins are associated with the extracellular matrix of many tissues in Torpedo. J Cell Biol. 1988;106:1263–1272. 78. Saarela J, Rehn M, Oikarinen A, et al. The short and long forms of type XVIII collagen show clear tissue specificities in their expression
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83. Aravind L, Koonin EV. The DNA-repair protein AlkB, EGL-9, and leprecan define new families of 2-oxoglutarate- and iron-dependent dioxygenases. Genome Biol. 2001;2:0007.1–0007.8. 84. Lauer M, Scruggs B, Chen S, et al. Leprecan distribution in the developing and adult kidney. Kidney Int. 2007;72:82–91. 85. Ghiselli G, Siracusa LD, Iozzo RV. Complete cDNA cloning, genomic organization, chromosomal assignment, functional characterization of the promoter, and expression of the murine Bamacan gene. J Biol Chem. 1999;274:17384–17393. 86. Wu RR, Couchman JR. cDNA cloning of the basement membrane chondroitin sulfate proteoglycan core protein, bamacan: a five domain structure including coiled-coil motifs. J Cell Biol. 1997;136:433–444.
Endocrinology
33 Sebum Production Claudine Pie´rard-Franchimont . Pascale Quatresooz . Ge´rald E. Pie´rard
Introduction Sebum is produced exclusively by the sebaceous glands. It serves as a vehicle for odors involved in sexual and social attraction. By a similar mechanism, the newborn child commonly recognizes his/her mother’s body odor. The reciprocal recognition is likely during the first weeks of life when the sebaceous glands are active in the newborn. It is noteworthy that the individual sebum-driven scents of each human being are commonly detected by dogs on skin and clothes. Other volatile compounds corresponding to pheromones are produced by mammalian skin in a mixture of apocrine sweat and sebum. In addition, sebum brings vitamin E, the melanocyte-stimulating hormone isotype a (a-MSH), and other various compounds to the stratum corneum (SC). Sebum interferes with the skin microbial biocenosis. It is fungistatic to dermatophyte species. Tinea capitis caused by Microsporum or Trichophyton spp typically occurs only before puberty when sebum production is minimal to absent. In addition, sebum exhibits some bacteriostatic properties. It promotes the growth of specific anaerobic and lipophilic microorganisms and parasites including Propionibacterium spp, Staphylococcus epidermidis, Malassezia spp, and Demodex mites. Other fields of biology are influenced by sebum. The high squalene content in sebum supposedly constitutes, after resorption, a substrate for cholesterol and vitamin D synthesis by the epidermis. Plasticity and cohesion between corneocytes are somewhat related to the sebum amount. Sebum protects the skin and the hair shaft from damage induced by mild acid solutions and friction. Indeed, combing and hairdressing generate friction effects on the cuticlar cells. Skin and hair surfaces are primarily hydrophobic. They paradoxically become more wettable following deposit of sebum components including free fatty acids [1]. In fact, surface wettability is involved in various protective functions of the SC including biocenosis preservation, smoothness, resiliency, and barrier effect to various xenobiotics. Sebum production shows large interindividual differences. Nonetheless, there are global influences of age and gender. In adults, the critical period in life regarding sebum production begins after menopause and andropause.
In older subjects, the sebum flow at the skin surface usually runs dry. From global perspective, aging of the sebaceous glands appears quite complex.
Seborrhea in a Cosmetic Perspective Sebum production shows large interindividual variations as well as intraindividual fluctuations with age. Any excess in the sebum output defines seborrhea that leads to unpleasant cosmetic aspects, and possibly fuels-specific disorders. Most seborrheic subjects exhibit both greasy hair and greasy skin on the forehead and nose. However, some relative differences may be found between these locations. After wiping or washing the skin, the sebum coating is often quickly restored. Hence, seborrhea commonly represents a matter of concern to the affected individual. Observing the skin under ultraviolet light discloses specific subclinical or faint patterns of epidermal melanization [2, 3]. Typically, a speckled perifollicular melanotic pattern (SPMP) is disclosed on the face and scalp of seborrheic individuals, in particular, those with androgenic alopecia [2]. SPMP is evidenced well before the development of other distinct patterns of photodamage-related melanosis. In addition, SPMP is absent in children when sebaceous glands are quiescent. It is also absent in the nonseborrheic parts of the body, including sun-exposed areas. It has been postulated that SPMP on seborrheic skin result from the melanocyte activation by a-MSH produced by the follicular infundibulum and present in sebum [2]. Greasy hair loses its natural luster, and it looks dull, darker, and moist. Hair shafts are weighted down with leaking sebum, which makes them adherent and flattens all hairstyles in thick masses on the scalp. They are difficult to comb. This condition is accompanied by adverse effects on hold. Sebum appears to have the same effect as a humid environment on hair hold. When feeling the oily tresses of sticky hairs, their limpness is perceptible and it is difficult to separate the individual hairs. Sebum is left on the fingers and clothes. The possibility to render a piece of paper translucent helps to distinguish sweaty and greasy hair. On heating, an aqueous impregnation disappears
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rapidly by evaporation, while lipids remain. It should be noted that sustained sweating may be associated with increased seborrhea. A scaly dermatitis is commonly present on seborrheic scalp. This condition is sometimes associated with pruritus or slight discomfort during the days preceding a shampoo. While these symptoms disappear with a regular shampoo, dandruff becomes more visible, since scales are no longer stuck to the scalp by sebum, and they are more easily shed on to the clothes and pillow.
Structure of the Sebaceous Gland The mature sebaceous glands are holocrine lobulated structures distributed all over the skin except on the palms and soles (> Fig. 33.1). Apart from specialized sites such as the eyelids and prepuce, sebaceous glands open indirectly at the skin surface via the hair follicle. The density of sebaceous glands differs from site to site on the body, being highest on the face and scalp followed by the back, chest, abdomen, arms, and legs. The face contains 300–1,500 glands/cm2, the scalp about 300–500 glands/cm2, and other sites present 100 glands/cm2 or less. Three distinct types of pilosebaceous follicles are identified according to the volume of the sebaceous glands and the size of the associated hairs. They are termed terminal hair follicle, vellus hair follicle, and sebaceous follicle, respectively. Terminal hair follicles are present on the scalp and beard region in men. The corresponding hair shaft is thick and the sebaceous gland is of medium to large size reaching about 1 mm3. Vellus hair is found over
. Figure 33.1 Sebaceous glands highlighted by an anti-EMA (epithelial membrane antigen) antibody
the entire body surface except in the palms, soles, and areas with terminal hair follicles. In vellus hair follicle, the hair shaft is short and thin, and the sebaceous gland is tiny when present. The sebaceous follicle is only found in humans. The gland is quite large, whereas the hair shaft is miniature and does not reach the skin surface. The holocrine process of sebum production begins with the proliferation of basaloid undifferentiated cells located at the periphery of the acini as well as in transglandular partitioning epithelial strands. During sebocyte maturation and lipid synthesis, cells enlarge up to 150fold in volume and they express the epithelial membrane antigen (EMA). They move toward the ostia of the glands. Finally, the cell wall ruptures, the lipid content and the cellular remnants form the sebum that is discharged into the sebaceous duct and further to the pilosebaceous infundibulum. The pilosebaceous infundibulum forms a reservoir, which may contain a sizable amount of sebum. The overall transit time of sebocytes takes about 2–3 weeks within the gland and a further week or so through the follicular reservoir before reaching the SC surface. The SC acts as a sponge trapping part of the sebum and eventually resorbing it [4, 5]. In these respects, the bulk of lipids present at the skin surface depends on so many variables that it does not directly reflect the metabolic events taking place in the glands themselves.
Lipid Composition of the Sebum Skin surface lipids originate from two distinct sources: the keratinizing epithelium and sebum. The composition of lipids from these two origins greatly differs. Native sebum is made of triglycerides, wax esters, squalene, and cholesterol esters (> Table 33.1). In mature sebocytes, vacuoles almost filling the cytoplasm contain two components, clearly distinctive on electron microscopy. One is opaque, cloudy, and osmium-positive, probably enriched in squalene. The other is translucent and osmium-negative reflecting the presence of saturated lipids. Synthesis of the various components of sebum involves two different pathways including (a) squalene synthesis following the classical mevalonate and farnesyl pyrophosphate route and (b) fatty acids and wax esters synthesis. Cholesterol is only present in trace amounts in native sebum because it is part of the structural compounds of the cell rather than to the sebum itself. Indeed sebocytes do not process the necessary enzymatic equipment to synthesize cholesterol from squalene. Wax esters or squalene are
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. Table 33.1 Average lipid composition of sebum (%) Component
Native sebum
Skin surface
Triglycerides
57
30–40
Wax esters
25
22–25
Squalene
15
12
Cholesterol esters
3
7
Diglycerides
0
2
Free fatty acids
0
16–25
Ceramides
0
2
generally a marker of sebum excretion helping to differentiate the sebum contribution from epidermal lipids. The fatty acids of the triglycerides are varied, corresponding to either saturated or unsaturated compounds branched or not. They show straight hydrocarbon chains with even or odd numbers of carbon atoms. Wax esters contain the longest chains, the C16 and C18 fatty acids being the most abundant. During its transit up to the skin surface, the sebum composition is altered by oxidative processes and biodegradation partly induced by specific microorganisms [6]. Indeed, triglyceride hydrolysis by bacterial lipases gives rise to free fatty acids, and mono- and diglycerides. At the skin surface, epidermal lipids are admixed with the sebum forming a spotty or continuous lipid film. In adults, the relative contribution of epidermal lipids over the SC is minimal in areas rich in sebaceous glands, although it affects the surface lipid composition on the limbs and trunk away from the midline. On the scalp and forehead, for example, epidermal lipids amount at 5–10 mg/cm2 of skin, whereas sebum is present at 100–700 mg/cm2. Quantitative variations in sebum production following drug intake (e.g., retinoids, antibiotics, etc.) are likely associated with subtle changes in its molecular composition, which in turn may alter the cornification of the infundibulum and be a key factor for comedogenesis [7, 8].
Sebum Excretion and Spreading Sebaceous gland dynamics involves four distinct and successive features: sebum production (a secretion rate function), storage (a volume function), skin surface output (a delivery rate function), and SC permeation (an influx rate function). The oily appearance of skin and hair results
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from an excess of sebum excretion, spreading and interacting with sweat, SC, and hair. On the scalp, sebum appears partly as discrete droplets emerging from follicular outlets, and partly as a surface coating. The droplets are unevenly spread on the hair. In seborrheic subjects, the whole hairshaft appears to be fully coated with sebum. On the skin surface, sebum is usually accumulated in the follicular funnels from where it flows out. Sebum permeates the superficial layers of SC, but a homogenous film is only found in seborrheic subjects. Sebum migration occurs between contiguous hairs or in a swatch, due to capillary forces. After degreasing hairs, the initial refatting rate reaches about 2–3.5 mm/min.
Methods of Sebum Excretion Measurement Subjective methods for evaluating skin and hair greasiness are available based on tactile and visual scales. Their correlation with an overall rating into five classes (very dry, dry, medium, greasy, and very greasy) is convenient. These assessments are useful for the appraisal of sebumcontrolling products. Objective measurements of the sebum excretion provide a better evidence of greasy skin and hair. Over the years, a wide variety of ingenious methods have been developed for measuring the amount of sebum excreted at skin surface and present on hair [1]. Indeed, scalp and hair sebum amounts must be distinguished because they differ significantly. Estimating the amount of scalp sebum needs a miniature sampling method, and hair must be shaved 24–30 h before measurement. The sebum amount present at the skin surface is conveniently measured in vivo using photometric assessment, and lipid-sensitive tapes. A reproducibility of about 10% and a sensitivity threshold in the 5 mg range of lipid amounts are usually considered to be satisfactory [1]. Sebum production and secretion are not measurable in the gland itself. In contrast, sebum excretion at the skin surface after its transit within the storage and delivery units corresponding to the infundibulum reservoir is routinely measured. Several major methods contributed over the years in the evaluation of certain parameters quantifying the sebum bulk and rheology. It should be kept in mind that components of the excreted sebum are partly trapped as any xenobiotic by the SC [6]. Hence, part of sebum is free inside the infundibulum and at the skin surface, while another part permeates onto the SC before eventually being metabolized and further resorbed.
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Photometric Method The basic principle of the photometric method relies on the fact that opalescent glass, sapphire plate, or lipid-sensitive tapes of a given opacity to light becomes translucent when their surfaces are coated with lipids. The photometric procedure is time-saving, highly reproducible, and does not require specially trained scientific staff. The Sebumeter1 SM810 (C + K Electronic, Cologne) is a commercially available device, which has gained popularity [1]. Sebum is absorbed into a piece of matted plastic strip of 0.1 mm thickness placed on a roller, which is manually rewound before each measurement. The probe is pressed under constant pressure against the skin surface. Pitfalls may arise from skin microrelief and roughness, which impair the close contact between the probe and the SC. After the probe has been in contact with skin surface for 30 s, measured by an internal timer, it is placed back into the main unit of the Sebumeter1. Transparency of the plastic film is measured by a photocell linked to a microprocessor after the emitted light has passed back and forth through the strip. It is acknowledged that the measures are in good agreement with the actual amount of lipids present on the strip. In fact, it is estimated that an average of about 40% of total skin surface lipids is absorbed with one sampling. The digital readout displayed as micrograms per square centimeters gives the estimated total amount of lipids on the skin. In order to get valid data, it is necessary to take several samples within a given area so as to avoid problems associated with the heterogeneity of sebaceous gland activity. However, the calculated amounts may be inaccurate when seborrhea is intense because there is a saturation effect of the plastic strip. The photometric method yields a single global estimate of the casual bulk of lipids present on a given surface of the skin at one point in time. The test area is large compared to the size of the ostia of sebaceous follicles. Thus, any difference between the activity of individual sebaceous follicles remains impossible to evaluate by this method. A few overactive sebaceous follicles releasing a large amount of sebum contribute to a disproportionate large effect on measurements.
Sebum-Sensitive Tape Techniques The method using standardized hydrophobic lipid-absorbent tapes relies on opaque, open-celled, microporous polymeric films [1, 8, 9]. It is considered as complementary to and as useful as the photometric method [1, 10, 11]. The tape material has to be affixed to the skin by gentle
pressure ensuring the elimination of any air bubbles. When the sebum-sensitive tape is placed on a skin area possibly moved by muscles, the investigator should periodically check that the uniform contact between tape and SC is maintained throughout the test. Two proprietary tapes are currently available. One type is the regular Sebutape1 (Cuderm Corporation, Dallas) characterized by the presence of an adhesive coat on one side of the tape designed to adhere tightly to the SC. Such adhesive coat likely impairs the swift penetration of sebum into the tape. The other type of lipid-sensitive tape is designed to be applied for only a very short time to the skin surface without interfacing any adhesive coating. These commercially available tapes are the Instant Sebutape1 (Cuderm Corporation, Dallas) and the Sebufix1 (C + K Electronic, Cologne). Depending on the study design, the skin may be prepared prior to the timed collection by removing sebum from the skin surface. The collection time should be determined according to the type of tape. With the regular Sebutape1, the adhesive interposed between the lipid-sensitive film and the skin is a limiting factor to the transfer of lipids. This may be of importance when the rate of sebum excretion is low and/or when the duration of the test is short. On the other hand, a saturation effect occurs on regular Sebutape1 when evaluating intense seborrhea during a test period beyond 1 h. The amount of sebum collected over uninterrupted successive hours is in fact lower than the addition of hourly sebum collections during a similar cumulative period of time. This is associated with confluence of lipid droplets and inaccuracy in identifying each spot as a single sebaceous follicle. These features are the main reasons why sebum samplings longer than 1 h should be avoided when using regular Sebutape1. When using one of the uncoated tapes, a contact time of 30 s or less is appropriate. During the procedure, each follicular outlet enriched in sebum pours out lipids, which fill pores of the tape rendering it focally transparent. The size of the clear spots is proportional to the amount of sebum delivered. The number of spots reflects the number of sebum-rich follicular ostia. These parameters are conveniently evaluated by visual inspection alone. Looking at samples against a black background in reflection mode results in a black and white pattern that can be assessed using an ordinal scale. The method allows to obtain a rough, but reasonable estimate of skin greasiness without requiring sophisticated equipment. Better quantitative assessments are achieved using computerized image analysis [9–11], which represents the most sensitive and accurate method (> Fig. 33.2)
Sebum Production
when offering the possibility of recording the number and size of individual spots and calculating the mean and total area of spots (TAS). The free sebum content of follicular reservoir is conveniently assessed using Sebufix1 tapes affixed onto a recording video camera working under ultraviolet light illumination (Visioscan VC 981, C + K Electronic, Cologne). Computer-assisted image analysis provides proper readings [12]. A built-in microprocessor providing such information is present in the apparatus. A photometric evaluation obtained by measuring the intensity of light transmitted through the samples is an alternative rapid approach [13] and is roughly equivalent to the TAS value obtained by image analysis. A variant is represented by reflectance colorimetric assessment of the samples placed against a colored background [14]. However, these overall quantitative approaches lose one of the main benefits of the lipid-absorbent tape method, namely the evaluation of sebum excretion at each individual sebaceous follicle. A similar quantitative method was designed aiming at collecting data at any time during the tape application onto the skin. The basic principle relies on the measurement of the modifications in color of the tape that occur when it becomes transparent. It shifts the natural ‘‘white’’ color of the tape to a color closer to that of the skin itself. Reflectance colorimetry is conveniently expressed as DE*ab. The benefit of such an approach is the ability to obtain multiple measurements without removing the tape, and therefore to explore a continuous reading of the kinetics of sebum output. . Figure 33.2 Aspect of a computerized image from a lipid-sensitive tape. The black dots represent the sebum output at the follicular orifices
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There are some limitations to the proper interpretation of the data. Some are specifically related to the material itself. Sebum spots are subject to changes in their size and transparency depending on storage time and temperature. At 20 C or so, they should be evaluated at a defined time after removal, preferably within 24 h. When immediate evaluation is not possible, storage in a freezer at 30 C is advisable.
Combined Methods When using lipid-sensitive tapes alone, the interpretation of the number and size of lipid droplets with regard to the sebaceous glands may be uncertain. In fact, it is not valid to ascribe a single follicular outlet to each spot, particularly when the latter is large. In order to solve such an uncertainty, a two-step method was designed [9]. Before removing the sebum-sensitive tape from the skin, its outlines are delineated on the SC with an ink mark. In a second step, a follicular biopsy using a cyanoacrylatecoated polyester film (Melinex O, ICI Plastic division) is collected from that site. The follicular biopsy [15], which is an extension of the cyanoacrylate skin surface stripping conveniently, exhibits follicular casts and microcomedones [1, 3, 16]. The ink marks of the outlines of sebumsensitive film are harvested on this material. The skin surface stripping and the corresponding lipid-sensitive tape are then exactly superposed using the ink imprint as an adjusting mark. This dual material is examined under the microscope and processed in a computerized image analyzer (> Fig. 33.3). This method allows simultaneous . Figure 33.3 Cyanoacrylate skin surface stripping superposed to the corresponding lipid-sensitive tape. This method highlights the active sebaceous follicles contrasting with the inactive follicles
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assessment of the size of lipid droplets and that of the corresponding follicular ostia and microcomedones [10]. A surrogate method relies on examination using a video camera working under ultraviolet light illumination (Visioscan VC981, C + K Electronic, Cologne). A frame designed to precisely attach and locate the camera is first affixed onto the test site. The aspect of skin surface and follicular outlets is recorded. In a second step, a Sebufix1 is interposed between the SC and the camera. The picture of lipid droplets is recorded after a 45-s collection. The comparison of both pictures identifies sebum-poor and sebum-rich follicles [12].
Quantitative Parameters of Sebum Excretion
2.5 mg/cm2/min on the forehead. On the scalp, it varies from 0.1 to 0.8 mg/cm2/min. TAS values yielded by the lipid-sensitive tape method represent a surrogate of SER evaluations. As a rule, SER is roughly correlated with CL. A linear relationship is commonly yielded between four successive 1-h SER and TAS measurements at least in the medium range of severity degree of seborrhea. The correlation is lost when the sebum output is either very low or quite high. This finding indicates that these parameters are related to the delivery of the pool of sebum already secreted by the gland and stored in the outer portion of pilosebaceous duct. Thus, it is clear that an initial 3–4-h collection is a measurement of sebum excretion rather than sebum secretion.
Sebum Replacement Time
The various sampling procedures of sebum provide information about a series of specific parameters. As a rule, the values obtained for these parameters may differ from subject to subject by a tenfold coefficient, but each value is a representative of a given subject in a precise environment.
Sebum replacement time appears to be a cumbersome parameter difficult to manage. It refers to the time needed to recover CL after removing sebum from the skin surface. It has been reported to take about 4 h in subjects with sebum excretion in the medium range.
Sebum Casual Level
Density in Sebum-Enriched Reservoirs
Sebum casual level (CL) is defined as the amount of lipid present at equilibrium when the skin surface remains untouched for several hours. It is a global estimate of skin greasiness. For practical reasons, most researchers record CL after a 4-h lag time following uncontrolled removal of the sebum film from the skin surface. It is expected but uncertain that the measurement reflects a plateau value, and hence CL is not recommended as a single parameter for in-depth studies of the sebaceous system. Therefore, it is believed to be a constant value for each normal adult. In contrast, interindividual variations are large as shown by CL ranging from 100 to 700 mg/cm2 on the forehead of healthy subjects. Similar wide variations are found also on the scalp.
The number of spots over a lipid-sensitive tape is a rough indicator of the density of follicular reservoirs enriched in sebum. Such a figure is usually lower than the number of sebaceous glands present on that area of skin This information can be confirmed by staining cyanoacrylate follicular biopsies for lipids. The sebum delivery at a given follicular ostium may represent a clue for the presence of an actively secreting sebaceous gland. It may also represent the site of a follicular reservoir passively filled by the sebum coming from the skin surface. Using the combination of lipid-sensitive tape and follicular biopsy it was clear that many follicular ostia neither store sebum nor are a route for sebum outflow. It is also possible that one single droplet corresponds to merging of several smaller ones. The size of the follicular outlet at skin surface is not correlated with the presence or absence of sebum [10].
Sebum Excretion Rate Sebum excretion rate (SER) refers to the amount of sebum excreted on a given skin area during a defined period of time. The duration of the definitive collection period is important because SER progressively decreases over the first hours after degreasing the skin. SER of the first hour sampling usually ranges from 0.5 to
Instant Sebum Delivery SER and TAS decrease almost linearly during the first hours of collection time. Calculating the regression line for the cumulative data at 1 h intervals allows to extrapolate a theoretical value for instant sebum delivery (ISD) at
Sebum Production
T0. This parameter supposedly reflects the spontaneous leakage of free sebum from follicular reservoir. ISD is not always correlated with SER. Similar information to ISD is provided by the Sebufix1 tape applied to the skin for a few seconds.
Follicular Excretion Rate The slope of the above-mentioned regression line between cumulative TAS over time has been coined follicular excretion rate (FER). It is a measure of the delivery rate of sebum from the follicular reservoirs. FER is not infrequently related to the first hour TAS value although physiological influences and some topical compounds may interfere with such a relationship.
Factors Affecting Sebum Excretion Physicochemical Regulation SER and FER are influenced by the physicochemical characteristics of sebum. Environmental and skin temperatures, and the balance between the sebum molecular components affect the viscosity and rheology of lipids at the skin surface [17]. This could explain in part some chronobiological variations in SER and FER including seasonal fluctuations [18, 19] as well as the influence of the ovarian cycle [20, 21] and perhaps other chronobiological rhythms of unknown periodicity. A circadian rhythm has been described with sebum output being optimal in the mid morning, and minimal during late evening and early morning hours [21]. The width of follicular ostium may greatly influence sebum rheology. There is an inverse relationship between the fluid flux and the fourth power of the radius of the tube in which the fluid passes through. Hence, variations in corneocyte accumulation and swelling at the lips of follicular outlets, as may occur during the ovarian cycle or after occlusion may influence sebum rheology [20, 21]. Skin surface energy phenomena result from molecular interactions. They are involved in sebum and sweat dispersion. Sweating is indeed an important confounding factor in rating sebum excretion. Even in dry skinned subjects, intensive or continuous sweating increases the CL. The sebum of seborrheic subjects appears as an oily and homogeneous fluid barely emulsified with water at ordinary temperatures. However, in some instances, it can be emulsified with sweat though it may take several hours.
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The theory of a continuous sebum excretion is opposed to the concept of a discontinuous excretion with a feedback control by the CL. SER appears to decline progressively as CL regains its initial value. This observation supported the hypothesis of a feedback mechanism controlling sebaceous excretion by the lipid film developed on skin surface. This concept was further strengthened by the fact that the amounts of sebum collected at constant time intervals seem to increase with the number of degreasing procedures. Sebum excretion stops spontaneously even in highly seborrheic subjects if the area, although isolated, remains uncovered. However, the plateau effect in excretion kinetics is only apparent and due to the spreading of sebum over a larger surface, or to its permeation into the upper layers of the SC. It is concluded that the initial phase of sebaceous secretion is likely continuous or fluctuating as a result of the combination of chronobiological rhythms. Any feedback mechanism from CL could only affect the sebum excretion thus modifying sebum storage rather than sebum production. Shampoos dedicated to greasy hair are commonly used as sebum-controlling products. Some chitosanderived molecules hinder sebum coating of the hairshaft. Particles of dry shampoos adhere to hair, retain lipids, and exert electrostatic forces repulsing the hairshafts. Frequent regular shampoos do not increase the sebum output on the scalp and do not influence the sebum coating of hair. In contrast, cationic polymers and silicone oils used in some shampoos facilitate sebum spreading. Regreasing studies on scalp and hair show general differences according to the level of greasiness. Hair sebum amount is typically lower than scalp sebum. Scalp CL recovers completely after 1–4 days following hair washing, at least for greasy hair types [22] and the aspect remains fairly constant in the following days. Scalp SER has been reported to progressively increase until 24 h after shampooing when it reaches its maximum value.
Hormonal Regulation Human skin, in particular the sebaceous glands, receive, produce, and coordinate hormone activation and inactivation through various molecular signals. The involved physiological mechanisms belong to the endocrine, paracrine, juxtacrine, autocrine, and intracrine hormonal repertoire. Free testosterone and 3a-dihydrotestosterone (DHT) are considered to exert a major boosting and dose-related effect on sebocyte proliferation and sebum secretion in
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man [23]. The levels of type 1 isoform of 5a-reductase are significantly higher in sebaceous glands than in other skin structures [23, 24]. Cells of the infundibulum are also reported to be sensitive to the same hormones. In women, the most important androgen is D4-androstenedione, which is produced by adrenal gland and ovaries. It may be converted into testosterone, but it possesses some intrinsic androgenic activity. 5a-androstane-3b, 17b-diol is a potent androgen and is the main metabolite of testosterone in back and scalp skin. Other androgen precursors of purely adrenal origin such as dehydroepiandrosterone (DHEA) explain sebaceous development in the fetus, after birth, and in the prepubertal years. DHEA can be converted into androstenedione and testosterone inside the sebocytes. By contrast, conversion of DHEA sulfate to DHEA only occurs with the assistance of monocytes exhibiting steroid sulfatase activity. The amount of circulating androgens is important to consider. However, the local production of sexual steroids provides autonomous control adjusting sexual steroid metabolism according to the body area. Facial and scalp sebocytes are particularly involved in this mechanism. The glucocorticoid receptor is another nuclear steroid receptor present in sebocytes. Glucocorticoids have been reported to stimulate sebocyte proliferation [23]. The second group of nuclear receptors, namely the thyroid receptor family, encompasses different soluble receptors in sebocytes. They correspond to the thyroid hormone receptor isotype b1, the estrogen receptor b, the retinoic acid receptors (RAR) isotypes a and g, retinoid X receptors (RXR) isotypes a, b, and g, and the peroxisome proliferator-related receptors (PPAR) isotypes a, d, and g. PPAR ligands augment the androgen stimulation of sebocyte differentiation [25]. Estrogens exert the opposite effect of androgens but with a much weaker potency. However, any decrease in size and excretion of sebaceous glands is only achieved with high doses of estrogens that are nonphysiological in women and feminizing in men. It is thus unlikely that normal estrogen levels play a role in inhibiting sebaceous gland activity. Following estrogen suppression, administration of testosterone restores sebum secretion. Estrogens at sebum-suppressive doses have been shown to reduce plasma and urine levels of testosterone. They also inhibit gonadotropin-releasing hormone synthesis and 5 a˜ reductase activity. There is no relationship between the growth rate of vellus hairs and sebum excretion. In contrast, the intensity of seborrhea and severity of evolving androgenic alopecia are often related. In this condition, GH and IGF-1 might play a role.
Neuropeptide Regulation A neurovegetative nerve plexus surrounds the sebaceous gland, but acetylcholine and adrenaline do not seem to influence sebum secretion. Sympathectomy has no effect on the level of surface lipids. A localized neurological lesion may conversely induce seborrhea in the involved site. Several types of neuropeptide receptors are present in sebocytes. They include the m-opiate receptor which bind b-endorphin, the vasoactive intestinal polypeptide (VIP) receptor, the neuropeptide Y receptor, and the calcitonin gene-related peptide (CGRP) receptor. Psychotropic drugs, often dopamine inhibitors, increase sebaceous secretion considerably. The same observation is found in Parkinson disease [26] perhaps due to high a-MSH serum levels. During treatment with levodopa, seborrhea decreases, but the drug is inactive on sebaceous excretion in normal subjects.
Ethnic, Gender, and Age Effects Some specific attributes related to sebum excretion have been ascribed to ethnic groups [27]. Such observation awaits for confirmation. As a consequence of the diversity of hormonal signals, sebum excretion varies according to age, gender, and climacteric period [28–30]. At any given age in men and women, both sebum excretion and secretion rates differ between individuals over a wide range. In addition, there is a huge overlap between data gained in both genders. Hence, it is not the amount of circulating androgens but rather the receptivity of the target tissues that accounts for interindividual differences in sebum excretion. It is clear that additional factors are likely to be involved. SER and FER values remain high in men until the eighth decade. In women, the rates remain unchanged until menopause. During the climacteric period, seborrhea may either increase or steadily decrease with age. Using the lipid-sensitive tapes, it is possible to evidence distinct patterns according to age and physiopathological conditions [27]. The size of follicular reservoirs and pores shows no tendency to shrink with age. A series of endocrine imbalances and a few drug treatments aiming or not at direct or indirect hormonal effects affect the activity of sebaceous apparatus. The most potent inhibitor of sebum excretion is the synthetic retinoid 13-cis-retinoic acid or isotretinoin, which at oral doses of 0.1–1 mg/kg/day inhibits sebum production by up to 80% within 6–8 weeks. Isotretinoin reduces cell renewal and lipid formation in the sebaceous gland.
Sebum Production
Isotretinoin reduces not only SER, but also the follicular reservoir, and both remain significantly suppressed for up to 1 year after therapy. Topical products are far less potent although of cosmetic interest [31, 32].
Conclusion The current knowledge of sebaceous gland physiology and sebum rheology on skin, scalp, and hair shaft has made some progress, leading to the introduction of new antiseborrheic agents. This breakthrough was made possible through the development of qualitative and quantitative methods for measuring the amounts of excreted sebum, and evaluating its lipid composition. Seborrhea on the scalp or forehead may represent a single phenomenon, or be part of a more complex system of multiple disturbances. As an interesting index of neuroendocrine physiology, it plays an increasing role in the understanding of biology, mainly due to the reliability of its measurement. The sebum flow is altered with aging. In any study in this field ethnicity, age range, gender, adequate skin profile, and test area on the body must be appropriate for a relevant design. The environmental conditions including seasons, relative humidity, and temperature should be controlled. In addition, skin temperature affects sebum rheology. A number of other physiological parameters modulate sebum excretion and some of them are responsible for chronobiological variations. Despite clever experimental designs, it should be stressed that panelists, perception, clinical gradings, and biometrological measurements are not always matched. Several objective methods have been devised for measuring the greasiness of skin, most of which involve the collection of sebum once it runs off the sebaceous apparatus. Preconditioning the skin by prior removal of sebum from the skin surface is a common procedure. Part of the sebum present inside the follicular reservoir can also be ignored by measurements. Uncontrolled depletion of the sebum pool impedes collection of reliable data. Conceptually, aging of the sebum production in women begins in the climacteric period after menopause. It is manifest in men well after entering the andropause period. In both genders, the early changes are often erratic and they progressively evolve to a reduction of the sebum flow.
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References 1. Pie´rard GE, Pie´rard-Franchimont C, Marks R, Paye M, Rogiers V. EEMCO guidance for the in vivo assessment of skin greasiness. Skin Pharmacol Appl Physiol. 2000;13:372–389. 2. Petit L, Pie´rard-Franchimont C, Saint Le´ger D, Loussouarn G, Pie´rard GE. Subclinical speckled perifollicular melanosis of the scalp. Eur J Dermatol. 2002;12:565–568. 3. Uhoda E, Pie´rard-Franchimont C, Petit L, Pie´rard GE. The conundrum of skin pores in dermocosmetology. Dermatology. 2005;210:3–7. 4. Blanc D, Saint Le´ger D, Brandt J, Constans S, Agache P. An original procedure for quantification of cutaneous resorption of sebum. Arch Dermatol Res. 1989;281:346–350. 5. Pie´rard-Franchimont C, Martalo O, Richard A, Rougier A, Pie´rard GE. Sebum rheology evaluated by two methods in vivo. Split-face study of the effect of a cosmetic formulation. Eur J Dermatol. 1999;9:455–457. 6. Saint-Le´ger D, Bague A, Lefebvre E, Cohen E, Chivot M. A possible role for squalene oxides in skin surface and intra-comedonal lipids of acne patients. Br J Dermatol. 1986;114:543–552. 7. Pie´rard-Franchimont C, Henry F, Fraiture AL, Fumal I, Pie´rard GE. Split face clinical and bio-instrumental comparison of 0.1% adapalene and 0.05% tretinoin in facial acne. Dermatology. 1999;198:218–222. 8. Nordstro¨m KM, Schmus HG, McGinley KJ, Leyden JJ. Measurement of sebum output using a lipid absorbent tape. J Invest Dermatol. 1986;87:260–263. 9. Pie´rard GE. Follicle to follicle heterogeneity of sebum excretion. Dermatologica. 1986;173:61–65. 10. Pie´rard GE. Rate and topography of follicular sebum excretion. Dermatologica. 1987;175:280–283. 11. Pagnoni A, Kligman AM, El Gammal S, Popp C, Stoudemayer T, et al. An improved procedure for quantitative analysis of sebum production using Sebutape. J Soc Cosmet Chem. 1994;45:221–225. 12. Pie´rard-Franchimont C, Pie´rard GE. Post-menopausal aging of the sebaceous follicle. A comparison between women receiving or not hormone replacement therapy. Dermatology. 2002;204:17–22. 13. Pie´rard GE, Pie´rard-Franchimont C. Effect of topical erythromycinzinc formulation on sebum delivery. Evaluation by a combined photometric multistep samplings with Sebutape. Clin Exp Dermatol. 1993;18:410–413. 14. Pie´rard GE, Pie´rard-Franchimont C, Kligman A. Kinetics of sebum excretion evaluated by the Sebutape-Chromameter technique. Skin Pharmacol. 1993;6:38–40. 15. Mills OH, Kligman AM. The follicular biopsy. Dermatologica. 1983;167:57–63. 16. Pie´rard GE, Pie´rard-Franchimont C, Goffin V. Digital image analysis of microcomedones. Dermatology. 1995;190:99–103. 17. El Khyat A, Mavon A, Leduc M, Agache P, Humbert P. Skin critical surface tension. A way to access the skin wettability quantitatively. Skin Res Technol. 1996;2:91–96. 18. Pie´rard-Franchimont C, Pie´rard GE, Kligman A. Seasonal modulation of the sebum excretion. Dermatologica. 1990;181:21–22. 19. Youn SW, Na JI, Choi SY, Huh CH, Park KC. Regional and seasonal variations in facial sebum secretions: a proposal for the definition of combination skin tpe. Skin Res Technol. 2005;11:189–195.
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20. Pie´rard-Franchimont C, Pie´rard GE, Kligman A. Rhythm of sebum excretion during the menstrual cycle. Dermatologica. 1991;182:211–213. 21. Verschoore M, Poncet M, Krebs B, Ortonne JP. Circadian variations in the number of actively secreting sebaceous follicles and androgen circadian rhythms. Chronobiol Int. 1993;10:349–359. 22. Black D, Lagarde JM, Auzoux C, Gall Y. An improved method for the measurement of scalp sebum. Curr Probl Dermatol. 1998;26:61–68. 23. Thiboutot D, Harris G, Iles V, Cimis G, Gilliland K, Hagari S. Activity of the type 1-5 alpha-reductase exhibits regional differences in isolated sebaceous glands and whole skin. J Invest Dermatol. 1995;105:209–214. 24. Zouboulis CC, Xia L, Akamatsu H, Seltmann H, Fritsch M, Hornemann S, et al. The human sebocyte culture model provides new insights into development and management of seborrhoea and acne. Dermatology. 1998;196:21–31. 25. Rosenfield RL, Deplewski D, Kentsis A, Ciletti N. Mechanisms of androgen induction of sebocyte differentiation. Dermatology. 1998;196:43–46. 26. Martignoni E, Godi L, Pacchetti C, Berardesca E, Vignoli GP, Albani G, et al. Is seborrhea a sign of autonomic impairment in Parkinson’s disease? J Neural Transm. 1997;104:1295–1304.
27. Nouveau-Richard S, Zhu W, Li YH, Zhang YZ, Yang FZ, Yang ZL, et al. Oily skin: specific features in Chinese women. Skin Res Technol. 2007;13:43–48. 28. Pie´rard GE, Pie´rard-Franchimont C, Leˆ T, Lapie`re C. Patterns of follicular sebum excretion rate during lifetime. Arch Dermatol Res. 1987;279:S104–S107. 29. Callens A, Vaillant L, Lecomte P, Berson M, Gall Y, Lorette G. Does hormonal skin aging exist? A study of the influence of different hormone therapy regimens on the skin of postmenopausal women using non invasive measurement techniques. Dermatology. 1996;193:289–294. 30. Caisey L, Gubanova E, Camus C, Lapatina N, Smetnik V, Le´veˆque JL. Influence of age and hormone replacement therapy on the functional properties of the lips. Skin Res Technol. 2008;14:220–225. 31. Pie´rard GE, Cauwenbergh G. Modulation of sebum excretion from the follicular reservoir by a dichlorophenyl-imidazoldioxolan. Int J Cosmet Sci. 1996;18:219–228. 32. Pie´rard GE, Ries G, Cauwenbergh G. New insight in the topical management of excessive sebum flow at the skin surface. Dermatology. 1998;196:126–129.
5 Skin Aging: A Brief Summary of Characteristic Changes Christina Raschke . Peter Elsner
Introduction In the last few decades, life expectancy in many industrialized countries has consistently increased and is predicted to be a continuing process [1]. Appropriate care of elderly skin gains increasing medical importance. Since agedependent effects are so manifest in skin appearance, structure, mechanics and barrier function, much effort has been placed in research to better understand them. Aging is a consequence of genetic program and cumulative environmental damage, which contributes to a progressive loss of structural integrity and physiological tasks of the skin [2, 3]. The intrinsic aging (physiological-, chronological-, UV-protected aging) of the skin is determined by genetic influences and internal factors such as hormones or metabolic substances [4]. Intrinsic aging is mainly controlled by progressive telomere shortening [5]. Telomeres, situated at the ends of eukaryotic chromosomes, are repeated DNA sequences that undergo a base-pair loss during DNA replication. Telomere shortening acts as a mitotic clock, leading to replicative senescence. The clinical aspect of intrinsically aged skin is visible on the consistently cloth-covered skin areas of elderly persons. Extrinsic aging is a similar process that superimposes on the process of intrinsic aging. It is caused by exogenous factors such as ultraviolet (UV) radiation, environmental toxins and infectious agents that induce DNA alterations and damage the skin [6]. The most important extrinsic factor inducing preterm skin aging is ultraviolet irradiation, which causes photoaging of the skin. UV irradiation accelerates telomere shortening in sun-exposed skin [5]. UV-induced extrinsic aging is visible on chronically UV-exposed skin areas in persons frequently engaged in outdoor activities. Acute and chronic sun exposure induces short-term and long-term effects on the skin, ranging from sunburn (erythema) and suntan up to the development of skin aging and skin cancer. Besides natural sunlight there is an increasing influence of artificial UV radiation (solarium). The long-wave UVA radiation (320–400 nm) enters the deep dermis, while the more energetic UVB light (290–320 nm) is absorbed mainly in
the epidermis, especially in the keratinocytes and melanocytes [7]. The direct interaction of UVB with cellular DNA induces damage of DNA strands [8]. UVA radiation also damages the DNA but less than UVB radiation. UVA damage is induced indirectly, through absorption by other endogenous chromophores that release reactive oxygen species, resulting in lipid peroxidation, activation of transcription factors and formation of DNA-strand breaks [8, 9]. Age-related physiological changes in elderly skin include clinical, histological, and biochemical changes, as well as changes in neurosensory perception, barrier function, wound healing and a higher incidence of benign and (pre-)cancerous diseases. Several lines of evidence suggest that the processes of intrinsic and extrinsic aging have different biologic mechanisms.
Clinical Aspects of Aging Skin The most obvious clinical impressions of the elderly skin are increased formation of wrinkles and deficits in elasticity (> Fig. 5.1). Functional changes in the skin of elderly persons include a decreased growth rate of the epidermis, hair and nails. The skin of the elderly is characterized by a decreased lipid content with a changed composition of lipids. These changes induce a rough and dry surface of the skin with tendency to irritation and redness and are likely to contribute to the increased susceptibility of aged skin to the disruption of barrier function [10]. Decrease of barrier function in the elderly skin alters penetration of contactants and primes the inflammatory response [11]. The secretion of sebaceous and sweat glands is diminished [12]. The immune response is reduced and increases the risk of suffering from age-dependent diseases [13]. Increasing degeneration and disorganization of rarefied capillaries and small vessels in elderly skin induce changes in circulation and thermoregulatory functions with predisposition to hypothermia [14]. The elderly skin is increasingly vulnerable to environmental injury by decelerated wound healing and re-epithelization, resulting in a higher
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_5, # Springer-Verlag Berlin Heidelberg 2010
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Skin Aging: A Brief Summary of Characteristic Changes
. Figure 5.1 Obvious signs of aging skin (Courtesy: Department of Dermatology, Jena)
pigmentation inducing the clinical aspect of irregular pigmentation of so-called photo-induced age spots, hyperpigmentations and hypomelanosis guttata [18].
Biochemical Changes of Aging Skin
risk of surgical procedures. The susceptibility of the skin to harmful factors is dependent on creating effective repair mechanisms and inflammatory responses [15]. The intrinsically aged skin appears dry and pale and shows an atrophic aspect with fine wrinkles, which are due to the gravitational or conformational forces. Noticeable is a transparent image of the skin with a shining through of underlying vascular structures. Intrinsically aged skin displays a certain degree of laxity and a regular pigmentation and is prone to a variety of benign neoplasms. Clinically and morphologically, the extrinsically aged skin appears in the form of two types of photoaging with either an atrophic or rather a hyperproliferational aspect [7]. The atrophic form shows small wrinkles and numerous telangiectasias on skin areas with intensive sun exposure [7]. The hyperproliferational form is thicker with a leather-like image and deeper wrinkles. Favre–Racouchot syndrome is a type of photoaging with the clinical signs of deep furrowing, nodular elastic changes, comedones and keratinous cysts tending to appear in the periorbital region of the face [16]. Erythrosis interfollicularis colli, a further example for extrinsic skin aging, shows a restricted erythema sparing the hair follicles. Lentigines solaris and seniles located on the forearms and back of the hands are typical signs of photoaging [17]. Melanocytes along the basal membrane vary in reference to size, morphology and
The surface pH of the normal skin averages a slightly acid value of pH 5.5 and increases in the aged skin [3]. A rise in cutaneous pH value increases susceptibility suffering skin damage of infection, allergy and irritation [19]. The intrinsic aging is mainly associated with decreasing levels of several hormones. The coincidence of climacteric symptoms and the beginning of intrinsic skin aging suggests that estrogen seems to play a dominant role in this process [20]. Intrinsically aged skin displays a significantly reduced expression of the extracellular matrix protein (ECM-1) in lower and upper epidermal layers inducing potential changes of the normal skin structure and function [21]. Most aging changes, intrinsically and extrinsically caused, are due to molecular damage caused by free radicals [22]. The increase in UV irradiation on the earth due to stratospheric ozone depletion may increase the risk of photooxidative damage induced by the generation of reactive oxygen species. Free radicals are highly reactive due to possession of an unpaired electron. Trans-urocanic acid is a major chromophore for UV radiation in human epidermis that undergoes isomerization to its cis-isomer due to UV exposure [23]. Aerobic metabolism generates the superoxide radical, which is metabolized by superoxide dismutase to form hydrogen peroxide and oxygen [24]. Hydrogen peroxide can rapidly generate an extremely reactive hydroxyl radical that damages DNA, proteins and lipids (> Fig. 5.2). The ultraviolet light-induced reactive oxygen species lead to induction of the transcription factor activator protein-1 (AP-1) [25]. AP-1 induces upregulation of matrix metalloproteinases (MMPs) like collagenase-1 (MMP-1), stromelysin-1 (MMP-3), and gelatinase A (MMP-2), which specifically degrade connective tissue proteins such as collagen and elastin and indirectly inhibit the collagen synthesis in the skin, resulting in the obviously changed photodamaged skin [26–30]. Increasing age reduces the activity of tissue inhibitor of metalloproteinase-1 (TIMP-1), an inhibitor of many members of the MMPs [27]. The balance between collagen synthesis and degradation up to collagen deficiency differs between intrinsic and photoaged skin. The intrinsic aging of the skin seems to be mediated by a reduction in collagen synthesis while the damage of dermal connective tissue in extrinsically aged skin is more about the induction of matrix metalloproteinases [31]. Skin damage is mediated
Skin Aging: A Brief Summary of Characteristic Changes
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. Figure 5.2 UVA-induced generation of reactive oxygen species (Figure adapted with permission from Ziemer et al. [7])
by imperfect protection mechanisms against reactive oxygen species. The mutations of the DNA may accumulate irreversibly if repair mechanisms such as excision repair or using the enzyme photolyase fail to act [7]. The mutancy of mitochondrial DNA is multiple higher than nuclear DNA, because mitochondria do not contain any repair mechanism [32]. The synthesis of collagen types I and III, mainly structural components of the dermal connective tissue, is diminished in photoaged skin by a down-regulation of type I and III procollagen expression [33]. AP-1 has also been shown to negatively regulate type I procollagen gene expression [34]. There are lot of other environmental damages implicated in the process of extrinsic skin aging like tobacco smoke, infrared light and ozone. Tobacco smoke is an important inductor of preterm skin aging. Lahmann et al. reported significantly more MMP-1 mRNA in the skin of smokers than nonsmokers, while no difference was seen for the tissue inhibitor of metalloproteinase-1 [35].
Structural Changes of the Aging Skin Histological analysis is routinely used to diagnose structural changes of skin pathologies. Recent technical progress permits a more precise presentation of the human skin and allows detailed physiological insights into skin changes. Quantitative measurements by bioengineering allow the noninvasive studying of aging skin. Laser Doppler Velocimetry analyses the cutaneous circulation even in vessels of the deeper skin layers [36]. The optical coherence tomography is a pulsed ultrasound technique that enables the fast
analysis of the skin thickness [36, 37]. Confocal laser scanning microscopy and multiphoton laser scanning tomography are noninvasive methods for in vivo presentation of the human skin and generate horizontal images parallel to skin surface [36, 37]. Multiphoton laser scanning tomography allows analysis of skin pathologies by quantifying autofluorescent agents and presenting specific decay rates. Structurally, the intrinsically aged skin shows a thinning of all layers, the corneocytes are less adherent to one another and the dermo–epidermal junction shows a flattening aspect. Due to the reduced dermo–epidermal contact area, the involved layers may dissolve in each other, inducing an increased vulnerability of the skin with formation of ecchymosis [7]. Progressive decreases in melanocyte and Langerhans cell density occur [13, 38]. The dermis has an atrophic aspect with a loss of cells and extracellular matrix and shows a decline of the vascular network that occupies the dermal papillae [39, 40]. Dermal collagen becomes sparser and elastin is degraded slowly and accumulates in intrinsically aged skin [36]. In contrast, the extrinsically aged skin shows a completely different histological aspect (> Fig. 5.3). The epidermis becomes acanthotic and dysceratotic with a high proliferation index of keratinocytes. The epidermis of photoaged skin shows atypia and dysplasia of keratinocytes and melanocytes and a loss of Langerhans cells [41]. The number of hair follicles is reduced more than in intrinsically aged skin, inducing a thinning of the hair [7]. A loss of sebaceous and sweat glands induces increasing dryness of the skin with itching sensations [15]. The histological hallmark of dermal photodamage is the increased synthesis of abnormally structured elastin with its accumulation as elastotic
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Skin Aging: A Brief Summary of Characteristic Changes
material, termed solar or actinic elastosis. Dermal collagen becomes sparser, degenerated and changed in composition [42]. Other characteristics of extrinsically aged skin are the flattening of the dermis, a histiocyte-lymphocyte perivascular infiltrate and a loss of rete ridges [41]. Actually the dermal changes in elderly skin may be demonstrated by in vivo autofluorescence and second harmonic generation measurements using multiphoton laser scanning tomography [43].
Age-Dependent Skin Diseases The process of skin aging is not only a cosmetic problem, but also of medical-dermatological relevance due to a . Figure 5.3 Histological aspect of photoaged skin. The histological image of photoaged skin includes tight stratum corneum, discrete epidermal atypia, teleangiectasias and solar elastosis (Courtesy: Department of Dermatology, Jena)
higher incidence of age-dependent skin diseases such as bullous pemphigoid, erysipelas and herpes zoster (> Fig. 5.4). Many dermatoses of the elderly are presented completely different than in young persons. Herpes zoster is more frequently associated with severe neuralgiform pains, erysipelas appears prevalently in the absence of fever and atopic syndrome is appeared in the form of pruriginous eczema. Clinical aspects of the aging skin are pruritus senilis, age-dependent pemphigoid and atrophic balanitis and vulvitis. Intrinsically aged skin is characterized by a higher incidence of benign hyperproliferations of keratinocytes and capillary blood vessels such as seborrheic keratosis and senile hemangiomas [7]. One feature of photoaging is the higher occurrence of benign hyperproliferative skin lesions like lentigines solaris as well as seborrheic keratosis and senile hemangioma (> Fig. 5.5). Lentigines seniles are pigmented maculae in chronically sun-exposed skin, which are due to an increase in the melanin content inside keratinocytes and casual with melanotic hyperplasia [44]. There is strong evidence supporting the direct role of sunlight exposure in the development of skin cancers. The UV-induced free radicals damage the DNA with possible mutations in oncogenes and tumor-suppressor genes or activation of cytoplasmic signal transduction pathways that are related to growth differentiation, senescence, transformation and tissue degradation [45]. The matrix metalloproteinases have been associated with the process of skin aging and in particular are thought to be critical for tumor invasion and metastasis [46]. An important feature of extrinsically aged skin is the higher incidence of praecancerosa such as actinic keratosis or morbus bowen and malignoma-like basal cell carcinoma and squamous cell carcinoma (> Fig. 5.6). Black skin according to Fitzpatrick is less susceptible to sunburn, photoaging and skin carcinogenesis. A higher melanin content and a different melanosomal dispersion pattern in the epidermis are thought to be responsible for its
. Figure 5.4 Typical age-dependent skin diseases. (a) Bullous pemphigoid, (b) erysipela, (c) herpes zoster (Courtesy: Department of Dermatology, Jena)
Skin Aging: A Brief Summary of Characteristic Changes
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. Figure 5.5 Age-dependent skin tumors. (a) Seborrheic keratosis, (b) senile hemangioma, (c) lentigines solaris (Courtesy: Department of Dermatology, Jena)
. Figure 5.6 (Pre-)cancerous skin diseases. (a) Actinic keratosis, (b) basal cell carcinoma, (c) squamous cell carcinoma (Courtesy: Department of Dermatology, Jena)
more resistant behavior to UV light [47]. Sunscreens with high amounts of sun protection factors protect against solar dermatitis causing a possible decrease in the formation of UV-induced skin malignancies [48].
Conclusion Most changes that occur in the aging face are related to gravity working on skin. Exposure to sunlight hastens these changes and protection from the sun is the only proved way to delay them. Regular photoprotection by avoiding solarium, using sunscreen, which should provide broad-spectrum UVB and UVA coverage, or wearing
UV-protective clothing is an integral part in the management of photoaging. It is important to minimize sun exposure during the peak hours of 10 AM to 4 PM. Tobacco smoke is toxic to cells and should be avoided to prevent accelerating skin aging. The changes of the elderly skin are largely a normal part of aging, but may induce a higher risk for serious skin complications. An increasing part of the elderly population feels disturbed about the age-dependent uncomely signs of the skin and wish therapeutic intervention. Despite the significant improvement that can be achieved with most anti-aging therapies, no technique is perfect. Some patients may have unrealistic expectations and may be unsatisfied with the resulting outcome. However, rational behavior during the life is the
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only proved way delaying preterm uncomely signs of skin aging and may help prevent complications.
Cross-references > Degenerative
Changes in Aging Skin
References 1. Kligman AM, Koblenzer C. Demographics and psychological implications for the aging population. Dermatol Clin. 1997;15 (4):549–553. 2. Farage MA, et al. Structural characteristics of the aging skin: a review. Cutan Ocul Toxicol. 2007;26(4):343–357. 3. Farage MA, et al. Functional and physiological characteristics of the aging skin. Aging Clin Exp Res. 2008;20(3):195–200. 4. Vaillant L, Callens A. Hormone replacement treatment and skin aging. Therapie. 1996;51(1):67–70. 5. Kosmadaki MG, Gilchrest BA. The role of telomeres in skin aging/ photoaging. Micron. 2004;35(3):155–159. 6. Menon G, Ghadially R. Morphology of lipid alterations in the epidermis: a review. Microsc Res Tech. 1997;37(3):180–192. 7. Ziemer M, et al. Alterungsprozesse der haut und altersdermatosen. In: Wedding U, et al. (ed) Medizin des alterns und des alten menschen, 1st ed. Berlin: Verlag Hans Huber, Hogrefe AG, 2007, pp. 157–165. 8. Griffiths HR, et al. Molecular and cellular effects of ultraviolet light-induced genotoxicity. Crit Rev Clin Lab Sci. 1998;35(3): 189–237. 9. Berneburg M, et al. Photoaging of human skin. Photodermatol Photoimmunol Photomed. 2000;16(6):239–244. 10. Rogers J, et al. Stratum corneum lipids: the effect of ageing and the seasons. Arch Dermatol Res. 1996;288(12):765–770. 11. Ghadially R. Aging and the epidermal permeability barrier: implications for contact dermatitis. Am J Contact Dermat. 1998;9(3): 162–169. 12. Balin AK, Pratt LA. Physiological consequences of human skin aging. Cutis. 1989;43(5):431–436. 13. Fenske NA, Lober CW. Structural and functional changes of normal aging skin. J Am Acad Dermatol. 1986;15(4 Pt 1):571–585. 14. Horvath SM, Rochelle RD. Hypothermia in the aged. Environ Health Perspect. 1977;20:127–130. 15. Harvell JD, Maibach HI. Percutaneous absorption and inflammation in aged skin: a review. J Am Acad Dermatol. 1994;31(6):1015–1021. 16. Patterson WM, et al. Favre-racouchot disease. Int J Dermatol. 2004;43(3):167–169. 17. Schafer T, et al. The epidemiology of nevi and signs of skin aging in the adult general population: results of the kora-survey 2000. J Invest Dermatol. 2006;126(7):1490–1496. 18. Ortonne JP. Pigmentary changes of the ageing skin. Br J Dermatol. 1990;122(Suppl 35):21–28. 19. Fiers SA. Breaking the cycle: the etiology of incontinence dermatitis and evaluating and using skin care products. Ostomy Wound Manage. 1996;42(3):32–34, 36, 38–40, passim. 20. Brincat MP, et al. Estrogens and the skin. Climacteric. 2005; 8(2):110–123.
21. Sander CS, et al. Expression of extracellular matrix protein 1 (ECM1) in human skin is decreased by age and increased upon ultraviolet exposure. Br J Dermatol. 2006;154(2):218–224. 22. Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol. 1956;11(3):298–300. 23. Kaneko K, et al. Cis-urocanic acid initiates gene transcription in primary human keratinocytes. J Immunol. 2008;181(1):217–224. 24. Fridovich I. Superoxide dismutases. An adaptation to a paramagnetic gas. J Biol Chem. 1989;264(14):7761–7764. 25. Lo YY, Cruz TF. Involvement of reactive oxygen species in cytokine and growth factor induction of c-fos expression in chondrocytes. J Biol Chem. 1995;270(20):11727–11730. 26. Angel P, et al. Function and regulation of AP-1 subunits in skin physiology and pathology. Oncogene. 2001;20(19):2413–2423. 27. Hornebeck W. Down-regulation of tissue inhibitor of matrix metalloprotease-1 (TIMP-1) in aged human skin contributes to matrix degradation and impaired cell growth and survival. Pathol Biol (Paris). 2003;51(10):569–573. 28. Varani J, et al. Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin. J Invest Dermatol. 2000;114(3):480–486. 29. Karin M, et al. Ap-1 function and regulation. Curr Opin Cell Biol. 1997;9(2):240–246. 30. Fisher GJ, Voorhees JJ. Molecular mechanisms of photoaging and its prevention by retinoic acid: ultraviolet irradiation induces map kinase signal transduction cascades that induce ap-1-regulated matrix metalloproteinases that degrade human skin in vivo. J Invest Dermatol Symp Proc. 1998;3(1):61–68. 31. Chung JH, et al. Modulation of skin collagen metabolism in aged and photoaged human skin in vivo. J Invest Dermatol. 2001;117 (5):1218–1224. 32. Richter C. Oxidative damage to mitochondrial DNA and its relationship to ageing. Int J Biochem Cell Biol. 1995;27(7):647–653. 33. Talwar HS, et al. Reduced type I and type III procollagens in photodamaged adult human skin. J Invest Dermatol. 1995;105 (2):285–290. 34. Chung KY, et al. An ap-1 binding sequence is essential for regulation of the human alpha2(I) collagen (col1a2) promoter activity by transforming growth factor-beta. J Biol Chem. 1996;271(6):3272–3278. 35. Lahmann C, et al. Matrix metalloproteinase-1 and skin ageing in smokers. Lancet. 2001;357(9260):935–936. 36. Waller JM, Maibach HI. Age and skin structure and function, a quantitative approach (I): blood flow, pH, thickness, and ultrasound echogenicity. Skin Res Technol. 2005;11(4):221–235. 37. Neerken S, et al. Characterization of age-related effects in human skin: a comparative study that applies confocal laser scanning microscopy and optical coherence tomography. J Biomed Opt. 2004;9(2):274–281. 38. Fenske NA, Conard CB. Aging skin. Am Fam Physician. 1988;37 (2):219–230. 39. Yaar M, Gilchrest BA. Skin aging: postulated mechanisms and consequent changes in structure and function. Clin Geriatr Med. 2001;17(4):617–630, v. 40. Yaar M, et al. Fifty years of skin aging. J Invest Dermatol Symp Proc. 2002;7(1):51–58. 41. Kerscher M, et al. Physiologie der hautalterung. In: Effendy I, Kerscher M (eds) Haut und Alter, 1st ed. Stuttgart: Georg Thieme Verlag KG, 2005, pp. 3–10. 42. Schwartz E, et al. Collagen alterations in chronically sun-damaged human skin. Photochem Photobiol. 1993;58(6):841–844.
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46. Crawford HC, Matrisian LM. Tumor and stromal expression of matrix metalloproteinases and their role in tumor progression. Invasion Metastasis. 1994;14(1–6):234–245. 47. Rijken F, et al. Responses of black and white skin to solar-simulating radiation: Differences in DNA photodamage, infiltrating neutrophils, proteolytic enzymes induced, keratinocyte activation, and IL10 expression. J Invest Dermatol. 2004;122(6):1448–1455. 48. Elsner P, et al. Sun protection: possibilities and limitations. J Dtsch Dermatol Ges. 2005;3(Suppl 2):S40–44.
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Part 1
Basic Sciences
Histology
1 Skin Aging in Animal Models: Histological Perspective Tapan K. Bhattacharyya
"
‘‘What lies beneath this aging skin? The untold destruction stealthy creeping, The bones the organs the nerves, the brain, I am breathing, I am functioning, Am I living?’’ (Julie A. Crippin, 2008)
animal models from the mammalian kingdom. This review will explore whether intrinsic aging also affects skin histology in animal models in a comparable manner. The data are very limited, and were acquired through painstaking investigation by scientists and clinicians, dating back to several decades, using classical histological techniques; the reported quantitative data were often uncorroborated by statistical methodology.
Introduction Intrinsic aging of human skin has been a topic of interest to scientists and clinicians for over a century, and numerous studies have been published which document progressive anatomic changes of human skin with age, through various decades of human life. Human populations worldwide are living longer, and skin from older people becomes more susceptible to diseases and malformations, apart from being ravaged by environmental trauma like ultraviolet radiation. Along with diseases, natural age-induced changes of the integumentary system pose problems for the elderly population at large. The science of clinical dermatology documents hundreds of diseases of human skin, but scant attention has been paid to basic research in the microanatomy of aging skin. This is an area of research that also invokes biological and evolutionary interest about the mechanics of age-induced involution of various biological systems. Unfortunately, documentation about chronological aging of animal skin is rather limited. The main reason for this is the scant availability of animal models of skin aging with clear documentation regarding age or the stages of the life cycle. Animal husbandry for long-term maintenance of aging animals in a disease-free colony is a costly project, and is usually beyond the reach of an average laboratory. The aging human skin is sagging, with increased roughness and wrinkling, inelastic, with thinning and mottling, and histologically shows many signs of atrophy, such as epidermal thinning or abnormal collagen and elastic fibers. It is logical to ask whether similar or different conditions are observed when the analysis is extended to
The Human Scenario: Skin Aging Histology Morphologic changes of the human integument due to intrinsic aging caused by time-induced physiological changes are understandable, but can be altered due to personal and environmental factors, can vary in different anatomic sites, and are also linked to endocrine factors [1]. Despite these problems, several accounts of intrinsic aging of human skin have been published over the last few decades. Skin atrophy is marked only after the fifth decade of human life, and shows a plethora of histomorphologic changes include epidermal thinning, flattening of the dermal-epidermal junction, loss of melanocytes, and immunocompetent Langerhans cells. There are also dermal changes such as reduced fibroblast population and sebaceous glands. These histopathologic events have been reviewed recently [2]. Morphometric measurement of collagen fibers from stained human skin biopsies further showed that collagen fiber density started decreasing from around 30–40 years, with thinner and more spaced fibers [3]. Other investigators found no difference in epidermal or dermal thickness in a study of wound healing comparing skin from young and elderly volunteers [4]. The biochemical profile of collagen metabolism of human skin, however, changes with age. A steady decline in synthesis of hydroxyproline in human skin up to the fourth decade has been described [5]. Reports of dermal elastic fiber changes with age, including abnormalities and disintegration, have been
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_1, # Springer-Verlag Berlin Heidelberg 2010
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published by some authors. As per the study of VitellaroZuccarello et al. [3], elastic fiber distribution has a different aging pattern in men and women. While the density of elastic fibers in the papillary dermis is not modified as a function of age, in the reticular dermis of both sexes the fiber density increases in the first decade, followed by a drop only in the female. Biochemically, elastin biosynthesis is stable up to approximately the fourth decade of life [5]. Although reports by different investigators vary in detail due to different kinds of sampling and histological methods employed, a general trend of attrition of the dermal connective tissue fibers is noted as a correlate of the aging process. Histopathologic changes in the epidermis and dermis of human skin in sun-exposed and sun-protected areas have been reviewed by MonteiroRiviere [6].
Aging in Rodent Models Some accounts of histological and cellular kinetic changes throughout the life span or representative stages of rodent life span are available. Most of the studies were conducted on the hairless mouse, other strains of mice (CBA, C57131/6NNia, Balb/c), and rats (Wistar rat, Fischer 344). It is difficult to make a meaningful comparison between older studies related to skin aging histopathology, as different authors have reported data on both sexes of various mouse models, which were often based upon limited sample numbers. Some of these important articles lack statistical evaluation. However, some noteworthy findings from early literature on different rodent models are summarized here. The hairless mouse owes its hairlessness to a homozygous recessive genetic condition, and structural changes of skin accompanying development of hairlessness in this animal were described in the older literature. Age-related modifications in epidermal cell kinetics in this species were described by Iverson and Schjoelberg [7]. Using autoradiography, it was shown that epidermal cell proliferation increased from birth to approximately 20 weeks of age, and remained steady. This detailed study could not confirm whether epidermal thickness or cell proliferation rate decreases systematically with increasing age. On the other hand, Haratake et al. [8] presented data showing that the thickness of the epidermis in hairless mice decreases with intrinsic aging. There was also less incorporation of tritiated thymidine in the epidermis in older mice. The tritiated thymidine technique was used in mice up to 19 months of age, and the data revealed an age-dependent decline in the cell proliferation rate [9]. The data reported from Balb/c mice [10] seemed to
indicate epidermal atrophy with age. The epidermis was thinner, with smaller nuclei, in 20-month old animals compared to 2-month animals, although there was no decrease in mitotic activity and DNA labeling index. The loss of epidermal mass was related to a decrease in protein and RNA content of the epidermis. However, similar epidermal changes were not observed in a detailed study in young and old C57B1/6NNia mice [11]. In fact, the epidermis from the ear and footpad showed a statistically significant increase in thickness and augmented cell size. The index of labeling with tritiated thymidine showed no difference between young and old mice. Moreover, in C57BL/6N mice, the number of epidermal cell layers and the epidermal thickness remained constant from 1 to 22 months of age [12]. Most of the reported studies on mouse skin were mainly concerned with the aging effect on the epidermal cell size or cell kinetics, and scarce attention has been devoted to the morphologic changes of the dermal constituents in aging animals. CBA mouse skin was investigated in three age groups (1, 6, and 27 months) from animals procured from NIH colonies [13]. As the rate of skin aging differs in different areas of the body [11], samples were studied from the dorsal, ventral, and pinna skin, and the footpad of these young, young adult, and old animals. A negative linear effect of age on epidermal depth with a significant reduction in cell count (cell/mm), and pilosebaceous unit profiles in dorsal skin samples and footpad was observed. The sebaceous glands appeared atrophied with pycnotic nuclei (> Figs 1.1 and > 1.2). No consistent change in depth of the dermis or area fraction of collagen as determined by histomorphometry was noticed. The dermal elastic fibers in the dorsal skin and footpad showed proliferation in higher age groups in this mouse model. In a study of young and old C57/B16 mice from NIH colonies, decreased dermal cellularity and thickness and decreased epidermal proliferation has been reported [14]. The aging skin in the rat shows non-uniform patterns in different strains when it comes to epidermal and dermal thickness, and the age-associated changes seem to differ from the trend noted in murine species. In Sprague-Dawley rats, the foot epidermis was explored to determine age-related changes in cell kinetics using single pulse [3H]-thymidine labeling and the percent labeled mitosis technique [15], and led to the conclusion that there is a progressive decline in the rates of cell proliferation associated with age. However, these data were presented from rats only up to the age of 52 weeks. The authors of this article referred to five reports published earlier, which showed that rodent epidermal cell proliferation decreased in middle age, and then remained
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. Figure 1.1 Histological preparation of the dorsal skin in aging CBA mice in young (a), young adult (b), and old (c) animals, showing increasing atrophy of the epidermis and shrinkage of sebaceous follicles. Dermal elastic fibers can be seen in c. Verhoeff-van Giesen stain
. Figure 1.2 Error bar chart of epidermal width measurements in three groups of CBA mice
constant, or increased in senile animals. Skin from aging Wistar rats up to the age of 34 months was studied using histomorphometric analysis by Voros and Robert [16]. Average epidermal and dermal thickness did not show appreciable change with senility in this species. In aging
Fischer 344 rats, epidermal thickness remained constant from 3 months of age onward [12]. However, increasing values in epidermal depth and nuclear population were noted in the ventral and dorsal skin and foot plate skin samples from young, 1-year-old, and 2-year-old Fischer 344 rats [17, 18]. Earlier, Lapiere [19] commented that instead of becoming atrophic, rat skin increases in size, with more collagen, although the rate of increase is greatly diminished with aging. This is due to reduced collagen biosynthesis and increased degradation of macromolecules, but the balance between synthesis and degradation remains positive in rats. Aging changes in cells other than epidermal keratinocytes, such as melanocytes or cells of Langerhans, have also been documented in some studies. Ultraviolet radiation has important health consequences on the Langerhans cells of human skin. The numerical density of Langerhans cells in aging inbred mice was studied from epidermal sheets, and showed reduction when compared to that in young animals, although cutaneous immunoreactivity was not compromised [20]. Age-related neurodegenerative changes in peripheral nerves is a widespread
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phenomenon of clinical importance, and rat studies have attested to this inhibitory pattern. Age-associated loss in size of Meissner corpuscles, with smaller and disorganized axonal processes, was reported in the digital pads of mice aged to the maximum life expectancy [21]. Interpretation of morphological changes of aging skin has many limitations, as discreet biochemical changes underlying such alterations cannot be visualized. In contrast to sparsely available accounts of dermal histochemical or morphological transformations in relation to life history in laboratory models, biochemical studies showing quantitative changes in dermal glycosaminoglycans, hydroxyproline concentration, acid mucopolysaccharides, and skin collagen and elastin changes in aging mice, rats, rabbits, and hamsters have been published. Only a few papers are cited here [22, 23]. Oxidative damage to the lipids and DNA increases with age in Fischer 344 rats, which was studied by measuring antioxidant enzyme activity [24]. In the hairless mouse, however, skin aging was not accelerated due to decreased antioxidant capacity [25]. Such molecular changes of the skin in intrinsically aged laboratory animals can only be revealed by immunohistochemistry as more suitable antibodies become available for research.
Morphologic Changes with Aging in Other Species Some sporadic accounts have also been published relating to mammals other than commonly available laboratory rodent models. Age-induced reduction in sebaceous glands was described in sheep by Warren et al. [26]. Epidermal flattening, fewer hair follicles and sebaceous glands, and a decrease in melanocytes were age-related changes in the hairless dog [27]. Veterinary textbooks describe aging changes in skin of domesticated dogs and cats. Senile changes in the skin of old cats and dogs include alopecia, callus formation over pressure points, orthokeratic hyperkeratosis of the epidermis, and atrophied hair follicles [28]. Due to certain structural similarities with human skin, the pig skin model has been used in many experiments for studying responses to surgical and physiological manipulations [29], but no account is available on aging morphologic changes in this species.
Calorie Restriction (CR) and Skin Aging Calorie restriction (CR) can reverse age-associated alterations in many organs like the heart, the liver, and
the brain [30], and has been shown to exert beneficial effects on many skin disorders, although its morphologic effect on the aging skin has not been thoroughly explored. Whether CR can modify age-related histomorphologic features of skin in colony-raised rats was evaluated with morphometric procedures in Fischer 344 rats in two aforementioned studies [18, 19]. Three age groups (young, adult, old) of this strain belonging to ad libitum and CR feeding regimens were obtained from NIH colonies, and skin samples from the dorsum, footpad, and abdominal skin were analyzed with morphometric procedures to evaluate various skin compartments (thickness of stratum corneum, epidermis, dermis, fat layer, percentage fraction of dermal collagen, elastic fibers, fibroblast density, capillary profiles, and staining intensity of dermal glycosaminoglycans). The Fischer 344 rat showed many age-related skin changes, and these were prevented or delayed by CR, presumably due to metabolic alterations imposed by the dietary regimen (> Fig. 1.3). CR reduces cell proliferation in some tissues, with inhibited pace of DNA replication, and this makes those tissues less susceptible to DNA damage by carcinogens. Epidermal cell proliferation as quantified by immunohistochemistry was also correlated to age-related changes in epidermal thickness in these colony-raised Fischer 344 rats. Just as CR somewhat inhibited the trend of increasing epidermal width in aging rats, the keratinocyte proliferation rate as measured by staining of proliferating cell nuclear antigen (PCNA) was correspondingly lower in aging CR rats. This trend was observed in the epithelium of the dorsal skin as well as the foot plate (> Figs. 1.4 and > 1.5) [31]. In C57BL/6J mice, epidermal cell proliferation was reduced by CR and alternate day fasting regimens [32]. A recent study in SENCAR mice also shows that dietary calorie restriction may inhibit gene expression in skin tissues relevant to cancer risks [33]. CR has been reported to reduce the level of free radicals and prevent accumulation of advanced glycation products, and thus may be beneficial to aged skin. On the other hand, loss of subdermal adipose tissue stores, with restricted feeding, may accentuate fine wrinkles in human facial skin. CR can diminish subdermal adipose tissue [18]; therefore, its esthetic effect on the profile of the aging human face remains to be seen.
Conclusion Due to the ease of studying skin aging phenomena and age-associated progressive changes in short-lived mammals, published studies were mostly confined to
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. Figure 1.3 Bar graph representation of the dermis depth from Fischer 344 rats in CR study. Three age groups of ad libitum (AL) and calorie-restricted animals (CR) are represented
laboratory rodent models. Unfortunately, many earlier descriptions of the histopathologic changes in aging skin in such models did not consider old or senile specimens, and therefore lack a proper population sampling. Thus, the story is somewhat incomplete when compared to gerontologic cutaneous changes studied from skin biopsy samples of 90-year old human patients! It should be noted that rodent life span is also highly variable in different strains. Early literature has recorded that, among inbred mice strains used for aging research, a mean life span can vary from a low of 276 days in the AKR/J strain to a maximum of 799 days in the LP/J strain [34]. The lack of availability of suitably aged rodent models is a handicap to such research. This review, however, shows that the mouse model may be more suitable for documenting age-related histological changes that can be compared to human data, despite the caveat that comparing skin from laboratory-grown rodents with a short life span with the human skin assaulted by years of disease and environmental challenge is a rather risky endeavor. Histological deficits in intrinsically aged human skin affect the epidermis, dermal thickness, cellularity, and the elastic fiber system. Efforts should be made to search for analogous chronologic changes in animal models that can be utilized for studies aimed at rejuvenation of the aged skin. Similarities between human and murine skin have been reported in
many studies, making mouse skin a suitable material for studying inflammatory skin diseases. Apart from certain similarities in basic molecular and physiological processes between the mouse and the human [35], the mouse can be maintained under proper husbandry conditions, and may be a more suitable animal for studying the process of senescence of the integumentary system. Virtually no data are available on histological changes from intrinsic aging in other animals like the dog, the domestic pig, or the rabbit, although such species are extensively used for biomedical and physiological studies. Wrinkle formation is a vexing problem of aging human facial skin, and there is little information about skin wrinkles in any animal model at the senile state. Suitable models for studying the histophysiology of wrinkles or their reversal have not been reported, except for one study. Cross-breeding between the Wistar and the wild rat was reported to generate a rat model (the Ishibashi rat) in which skin aging, with wrinkles and furrows, appeared at 12 weeks. Wrinkle formation in this new model was due to a reduction in elastin and collagen contents of the aging skin [36]. This kind of experimental model would prove useful to define the underlying anatomic correlates of skin wrinkle formation, to study its profilometry, and to test the effect of topical anti-wrinkle products for its amelioration [37].
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. Figure 1.4 Representation of epidermal width and PCNA-index from foot plate in rats from CR study. Three age groups from AL and CR animals are represented
. Figure 1.5 PCNA staining in foot plate epidermis from adult (a) and old CR (b) rats. (a) CR rat, adult group, foot plate section. A broad epidermis with a thick cornified layer, and brown stained PCNA reactive cells in the basal layer can be observed. (b) CR rat, old group. The foot plate section shows regression of the epidermis and fewer PCNA positive cells
Skin Aging in Animal Models: Histological Perspective
In recent years, stem cells in skin have attracted a lot of scientific introspection due to their potential clinical application in wound healing, burns, and alopecia [38], and these cells can go through self-renewal and terminal differentiation, and may regulate tissue aging [39]. It remains to be determined whether stem cells in aging mouse skin can show temporal regression. In vitro studies have shown aged mouse epidermal keratinocytes can function comparably as those cells from young mice [40]. Keratin 15 (K15) promoter activity, which is specific for stem cells, was shown to be active in the hair follicle bulge in murine skin, and the basal epidermal expression of K15 gradually decreased with age [38]. On the other hand, in C57/BI6 mice from NIH colonies, despite a loss in dermal cellularity and thickness in the dorsal skin associated with skin aging, epidermal stem cells were maintained at normal levels throughout life [14]. Further research will elaborate how stem cells can respond to intrinsic aging in different mouse models, and whether they can be pharmacologically stimulated to reverse the process of cutaneous aging.
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Acknowledgment The kind support and encouragement provided by Dr. J. Regan Thomas, MD, Chairman, Department of Otolaryngology-Head & Neck Surgery, UIC is greatly appreciated. Some of the cited work was partly supported by the Bernstein grant from the AAFPRS foundation.
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