Molecular Pathology: The Molecular Basis of Human Disease

By William B. Coleman and Gregory J. Tsongalis

As the molecular basis of human disease becomes better characterized, and the implications for understanding the molecular basis of disease becomes realized through improved diagnostics and treatment, Molecular Pathology, Second Edition stands out as the most comprehensive textbook where molecular mechanisms represent the focus. It is uniquely concerned with the molecular basis of major human diseases and disease processes, presented in the context of traditional pathology, with implications for translational molecular medicine.

The Second Edition of Molecular Pathology has been thoroughly updated to reflect seven years of exponential changes in the fields of genetics, molecular, and cell biology which molecular pathology translates in the practice of molecular medicine. The textbook is intended to serve as a multi-use textbook that would be appropriate as a classroom teaching tool for biomedical graduate students, medical students, allied health students, and others (such as advanced undergraduates). Further, this textbook will be valuable for pathology residents and other postdoctoral fellows that desire to advance their understanding of molecular mechanisms of disease beyond what they learned in medical/graduate school. In addition, this textbook is useful as a reference book for practicing basic scientists and physician scientists that perform disease-related basic science and translational research, who require a ready information resource on the molecular basis of various human diseases and disease states.

• Explores the principles and practice of molecular pathology: molecular pathogenesis, molecular mechanisms of disease, and how the molecular pathogenesis of disease parallels the evolution of the disease
• Explains the practice of “molecular medicine and the translational aspects of molecular pathology
• Teaches from the perspective of “integrative systems biology
• Enhanced digital version included with purchase

• Language: English
• Publisher: Academic Press
• Release date: Nov 9, 2017
• ISBN: 9780128027875

Book preview

Molecular Pathology

The Molecular Basis of Human Disease

Second Edition

Editors

William B. Coleman

Gregory J. Tsongalis

Table of Contents

Cover image

Title page

Copyright

Dedication

List of Contributors

Preface

Acknowledgments

Chapter 1. Molecular Mechanisms of Cell Death

Introduction

Modes of Cell Death

Structural Features of Necrosis and Apoptosis

Cellular and Molecular Mechanisms Underlying Necrotic Cell Death

Pathways to Apoptosis

Mitochondria

Nucleus

Endoplasmic Reticulum

Lysosomes

Shared Pathways to Necrosis and Apoptosis

Programmed Necrosis

Concluding Remark

Chapter 2. Acute and Chronic Inflammation Induces Disease Pathogenesis

Introduction

Leukocyte Adhesion, Migration, and Activation

Acute Inflammation and Disease Pathogenesis

Pattern Recognition Receptors and Inflammatory Responses

Chronic Inflammation and Acquired Immune Responses

Tissue Remodeling During Acute and Chronic Inflammatory Disease

Chapter 3. Infection and Host Response

Introduction

Microbes and Hosts—Balance of Power?

The Structure of the Immune Response

Regulation of Immunity

Pathogen Strategies

Staphylococcus aureus: The Extracellular Battleground

Mycobacterium tuberculosis and the Macrophage

Herpes Simplex Virus: Taking Over

The African Trypanosome and Antibody Diversity: Dueling Genomes

HIV: The Immune Guerilla

Perspectives

Chapter 4. Neoplasia

Introduction

Cancer Statistics and Epidemiology

Classification of Neoplastic Diseases

Characteristics of Benign and Malignant Neoplasms

Clinical Aspects of Neoplasia

Chapter 5. Basic Concepts in Human Molecular Genetics

Introduction

Molecular Structure of DNA, DNA Transcription, and Protein Translation

Molecular Pathology and DNA Repair Mechanisms

Modes of Inheritance

Central Dogma and Rationale for Genetic Testing

Allelic Heterogeneity and Choice of Analytical Methodology

Conclusion

Chapter 6. The Human Genome: Understanding Human Disease in the Post-Genomic Era

Introduction

Structure and Organization of the Human Genome

Overview of the Human Genome Project

Impact of the Human Genome Project on the Identification of Disease-Related Genes

Sources of Variation in the Human Genome

Types of Genetic Diseases

Genetic Diseases and Cancer

Perspectives

Chapter 7. The Human Transcriptome: Implications for Understanding, Diagnosing, and Treating Human Disease

Introduction

Gene Expression Profiling—Early Attempts to Search for Candidate Genes Involved in Normal Physiological Processes and Pathogenesis

Preparation of Target RNA From Biological and Clinical Specimens

Transcriptome Analysis Based on Microarrays—Technical Aspects

Transcriptome Analysis Based on RNA Sequencing—Technical Aspects

Bioinformatics I—Basic Processing of Microarray and RNA-seq Data

Bioinformatics II—Exploration and Statistical Evaluation of Transcriptomics Data

Repositories for Transcriptome Data

Trancriptome Analysis—Applications in Basic Research and Translational Medicine

Perspectives

Chapter 8. The Human Epigenome—Implications for the Understanding of Human Disease

Introduction

Epigenetic Regulation of the Genome

Genomic Imprinting

Cancer Epigenetics

Human Disorders Associated With Epigenetics

Environment and the Epigenome

Chapter 9. Clinical Proteomics and Molecular Pathology

Understanding Cancer at the Molecular Level: An Evolving Frontier

Microdissection Technology Brings Molecular Analysis to the Tissue Level

Serum Proteomics: An Emerging Landscape for Early-Stage Cancer Detection

Chapter 10. Integrative Systems Biology

Introduction

Data Generation

Data Integration

Modeling Systems

Implications for Understanding Disease

Discussion

Chapter 11. Pathology: The Clinical Description of Human Disease

Introduction

Terms, Definitions, and Concepts

A Brief History of Approaches to Disease

Current Practice of Pathology

The Future of Diagnostic Pathology

Conclusion

Chapter 12. Understanding Molecular Pathogenesis: The Biological Basis of Human Disease and Implications for Improved Treatment of Human Disease

Introduction

Hepatitis C Virus Infection

Summary

Acute Myeloid Leukemia

Summary

Cystic Fibrosis

Summary

Chapter 13. Integration of Molecular and Cellular Pathogenesis: A Bioinformatics Approach

Introduction

Overview of Bioinformatics

Database Resources

Data Analysis

The Future of Bioinformatics

Chapter 14. Molecular Basis of Cardiovascular Disease

Introduction

General Molecular Principles of Cardiovascular Diseases

The Cells of Cardiovascular Organs

Atherosclerosis

Ischemic Heart Disease

Aneurysms

Vasculitis

Valvular Heart Disease

Cardiomyopathies

Lymphatic Circulation

Chapter 15. Molecular Basis of Hemostatic and Thrombotic Diseases

Introduction and Overview of Coagulation

Disorders of Soluble Clotting Factors

Disorders of Fibrinolysis

Disorders of Platelet Number or Function

The Thrombophilias

Chapter 16. Molecular Basis of Lymphoid and Myeloid Diseases

Introduction

Development of the Blood and Lymphoid Organs

Myeloid Disorders

Lymphocyte Disorders

Outlook for the Treatment of Leukemia

Chapter 17. Molecular Basis of Diseases of Immunity

Introduction

The Normal Immune System

Major Syndromes

The Hygiene Hypothesis

Chapter 18. Molecular Basis of Pulmonary Disease

Introduction

Neoplastic Lung and Pleural Diseases

Nonneoplastic Lung Disease

Interstitial Lung Diseases

Surfactant Dysfunction Diseases

Pulmonary Vascular Diseases

Chapter 19. Molecular Basis of Diseases of the Gastrointestinal Tract

Introduction

Gastric Cancer

Colorectal Cancer

Chapter 20. Molecular Basis of Liver Disease

Molecular Basis of Liver Development

Molecular Basis of Metabolic Zonation in the Liver

Molecular Basis of Liver Regeneration

Liver Stem Cells in Liver Health and Disease

Molecular Basis of Hepatocyte Death

Molecular Basis of Nonalcoholic Fatty Liver Disease

Molecular Basis of Alcoholic Liver Disease

Molecular Basis of Hepatic Fibrosis and Cirrhosis

Molecular Basis of Hepatic Tumors

Chapter 21. Molecular Basis of Diseases of the Exocrine Pancreas

Introduction

Acute Pancreatitis

Inflammation: Cause and Consequence of Acinar Cell Damage

Chronic and Hereditary Pancreatitis

Summary

Chapter 22. Molecular Basis of Diseases of the Endocrine System

Introduction

The Pituitary Gland

The Thyroid Gland

The Parathyroid Gland

The Adrenal Gland

Puberty

Chapter 23. Molecular Basis of Gynecologic Diseases

Introduction

Benign and Malignant Tumors of the Female Reproductive Tract

Disorders Related to Pregnancy

Chapter 24. Molecular Basis of Kidney Disease

Introduction

Clinical Manifestations

Diagnosis of Renal Disease

Specific Glomerular and Tubular Diseases

Tubulointerstitial Fibrosis

Conclusions

Chapter 25. Molecular Pathogenesis of Prostate Cancer

Introduction

Incidence and Etiology of Prostate Cancer

Genetic Contributions to Prostate Cancer Risk

Somatic Alterations in Gene Expression

Epigenetics

Advances in Mouse Models of Prostate Cancer

Conclusion

Chapter 26. Molecular Biology of Breast Cancer

Introduction

Histopathological Classification

Biomarkers

Gene Expression Profiling

Massively Parallel Sequencing

Conclusions

Chapter 27. Molecular Basis of Skin Disease

Introduction

Skin Diseases and Their Impact

Molecular Basis of Healthy Skin

Skin Development and Maintenance Provide New Insight Into the Molecular Mechanisms of Disease

Molecular Pathology of Mendelian Genetic Skin Disorders

Molecular Pathology of Common Inflammatory Skin Diseases

Skin Proteins as Targets for Inherited and Acquired Disorders

Molecular Pathology of Skin Cancer

Molecular Diagnosis of Skin Disease

New Molecular Mechanisms and Novel Therapies

Chapter 28. Molecular Basis of Bone Diseases

Introduction

Molecular Basis of Bone Modeling and Remodeling

Molecular Basis of Bone Disease Associated With Bone Matrix

Molecular Basis of Bone Disease Associated With Bone Resorption

Molecular Basis of Metabolic Bone Disease

Molecular Basis of Bone Infection and Inflammation

Molecular Basis of Bone Cancer

Molecular Basis of Bone Metastasis

Chapter 29. Molecular Basis of Diseases of the Nervous System

Anatomy of the Central Nervous System

Neurodevelopmental Disorders

Neurological Injury: Stroke, Neurodegeneration, and Toxicants

Neoplasia

Disorders of Myelin

Chapter 30. Molecular Diagnosis of Human Disease

Introduction

Regulatory Agencies and CLIA

Quality Assurance, Quality Control, and External Proficiency Testing

Method Validation

Clinical Utility

Molecular Laboratory Subspecialties

Chapter 31. Molecular Assessment of Human Diseases in the Clinical Laboratory

Introduction

Molecular Pathology Techniques

Clinical Applications

Emerging Technologies

Chapter 32. Pharmacogenomics and Personalized Medicine in the Treatment of Human Diseases

Introduction

Historical Perspective

Genotyping Technologies

PGx and Drug Metabolism

Drug–Drug Interactions

PGx and Drug Triansporters

PGx and Drug Targets

PGx Applied to Oncology

Targeted Therapies in Oncology

Challenges Encountered

Conclusion

Index

Copyright

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Dedication

The wealth of information contained in this textbook represents the culmination of innumerable small successes that emerged from the ceaseless pursuit of new knowledge by countless experimental pathologists working around the world on all aspects of human disease. Their ingenuity and hard work have dramatically advanced the field of molecular pathology over time and in particular in the last three decades. This book is a tribute to the dedication, diligence, and perseverance of the individuals who have contributed to the advancement of our understanding of the molecular basis of human disease, especially the graduate students, laboratory technicians, and postdoctoral fellows, whose efforts are so frequently taken for granted, whose accomplishments are so often unrecognized, and whose contributions are so quickly forgotten.

The second edition of Molecular Pathology: The Molecular Basis of Human Disease is dedicated to the memory of Oliver Smithies, DPhil, Weatherspoon Eminent Distinguished Professor of Pathology and Laboratory Medicine at the University of North Carolina School of Medicine (Chapel Hill, NC) who passed away on January 10, 2017 at the age of 91 years. Dr. Smithies was a cherished colleague to everyone at UNC and renowned for his gentle character, generous spirit, infectious curiosity, and passion for science. He was a friend to all and eager to help others succeed. Dr. Smithies was also a giant in the field of genetics who made numerous seminal discoveries over the course of his lifelong career (over 70  years at the bench). In 2007, he was a corecipient of the Nobel Prize for Physiology or Medicine for his contributions to the development of techniques for homologous recombination, which enable genetic modification of mammalian cells. These techniques provide the methodological foundation for engineered (transgenic and knockout) animal models of disease, which have been so valuable in the study of human diseases. Despite his tremendous accomplishments, status in the field, and numerous awards and honors, Dr. Smithies was unpretentious and approachable. We are proud to have known him for many years and for the example he provided for us and so many others as a distinguished and accomplished experimental pathologist and a genuinely good person. Even though he is gone, Dr. Smithies continues to inspire generations of scientists who were fortunate enough to have known him to do their best work.

We also dedicate the second edition of Molecular Pathology: The Molecular Basis of Human Disease to the many people who have played crucial roles in our successes. We thank our many scientific colleagues, past and present, for their camaraderie, collegiality, and support. We especially thank our scientific mentors for their example of research excellence. We are truly thankful for the positive working relationships and friendships that we have with our faculty colleagues. We also thank our students for teaching us more than we may have taught them. We thank our parents for believing in higher education, for encouragement through the years, and for helping our dreams into reality. We thank our brothers and sisters, and extended families, for the many years of love, friendship, and tolerance. We thank our wives, Monty and Nancy, for their unqualified love, unselfish support of our endeavors, understanding of our work ethic, and appreciation for what we do. Lastly, we give a special thanks to our children, Tess, Sophie, Pete, and Zoe. Their achievements and successes as young adults are a greater source of pride for us than our own accomplishments. As when they were children, we thank them for providing an unwavering bright spot in our lives, for their unbridled enthusiasm and boundless energy, and for giving us a million reasons to take an occasional day off from work just to have fun. Now that they are older, we cherish their friendship and value their companionship.

William B. Coleman

Gregory J. Tsongalis

List of Contributors

Philippe Aftimos, MD,     Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium

Hatem A. Azim Jr. MD, PhD,     Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium

Sheldon I. Bastacky, MD,     Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States

David O. Beenhouwer, MD

Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, CA, United States

Division of Infectious Diseases, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, CA, United States

Jaideep Behari, MD, PhD,     Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, University of Pittsburgh, School of Medicine, Pittsburgh, PA, United States

Joseph R. Biggs, PhD,     Department of Pathology and Division of Biological Sciences, University of California San Diego, La Jolla, CA, United States

Sheldon Campbell, MD, PhD,     Department of Laboratory Medicine, Yale School of Medicine, VA Connecticut Healthcare System, New Haven, CT, United States

Wai-Yee Chan, PhD,     School of Biomedical Sciences, Faculty of Medicine, Lo Kwee-Seong Integrated Biomedical Sciences Building, The Chinese University of Hong Kong, Shatin, Hong Kong SAR

William B. Coleman, PhD,     Department of Pathology and Laboratory Medicine, Curriculum in Toxicology, UNC Program in Translational Medicine, UNC Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC, United States

Massimiliano M. Corsi Romanelli, MD, PhD

Department of Biomedical Sciences for Health, Università degli Studi di Milano, Milan, Italy

U.O.C SMEL-1 Patologia Clinica IRCCS Policlinico San Donato, Milan, Italy

Robin D. Couch, PhD,     Department of Chemistry and Biochemistry, George Mason University, Manassas, VA, United States

Justin B. Davis, PhD,     Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, United States

Sophie J. Deharvengt, PhD,     Laboratory for Clinical Genomics and Advanced Technology (CGAT), Department of Pathology and Laboratory Medicine, Dartmouth Hitchcock Medical Center, Lebanon, NH, United States

Armando J. Del Portillo, MD, PhD,     Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, NY, United States

Virginia Espina, PhD, MT(ASCP),     Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, United States

Manel Esteller, MD, PhD

Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Catalonia, Spain

Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona (UB), L’Hospitalet, Catalonia, Spain

Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Catalonia, Spain

Carol F. Farver, MD,     Director, Pulmonary Pathology, Vice Chair for Education, Department of Anatomic Pathology, Cleveland Clinic Foundation, Cleveland, OH, United States

Michael D. Feldman, MD,     Professor of Pathology, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States

Susan L. Fink, MD, PhD,     University of Washington, Seattle, WA, United States

Margaret Flanagan, MD,     Department of Pathology, Stanford University, Palo Alto, CA, United States

Claudia Fredolini, PhD,     SciLifeLab, School of Biotechnology, KTH – Royal Institute of Technology, Solna, Sweden

William K. Funkhouser Jr. MD, PhD,     Department of Pathology and Lab Medicine, University of North Carolina School of Medicine, Chapel Hill, NC, United States

Matthias E. Futschik, PhD,     School of Biomedical & Healthcare Sciences, Plymouth University Peninsula Schools of Medicine and Dentistry, Plymouth, United Kingdom

Emanuela Galliera, PhD

Department of Biomedical, Surgical and Oral Sciences, Università degli Studi di Milano, Milan, Italy

IRCCS Galeazzi Orthopedic Institute, Milan, Italy

Avrum I. Gotlieb, MDCM, FRCPC,     Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Laboratory Medicine Program, University Health Network, Toronto, ON, Canada

Robert F. Hevner, MD, PhD,     Department of Neurological Surgery, Seattle Children’s Hospital Research Institute, Seattle, WA, United States

W. Edward Highsmith Jr. PhD,     Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, United States

Christopher Dirk Keene, MD, PhD,     Department of Pathology, University of Washington, Seattle, WA, United States

Nigel S. Key, MD,     Department of Medicine, Division of Hematology/Oncology, University of North Carolina, Chapel Hill, NC, United States

Christine M. Koellner, MS, CGC,     Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, United States

John J. Lemasters, MD, PhD,     Departments of Drug Discovery & Pharmaceutical Sciences and Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, SC, United States

Markus M. Lerch, MD,     Department of Internal Medicine A, Ernst-Moritz-Arndt-Universität Greifswald, Greifswald, Germany

Lance A. Liotta, PhD,     Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, United States

Youhua Liu, PhD,     Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States

Karen H. Lu, MD,     Department of Gynecologic Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, United States

Nicholas W. Lukacs, PhD,     Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, United States

Alice D. Ma, MD,     Department of Medicine, Division of Hematology/Oncology, University of North Carolina, Chapel Hill, NC, United States

Karlyn Martin, MD,     Department of Medicine, Division of Hematology/Oncology, University of North Carolina, Chapel Hill, NC, United States

Julia Mayerle, MD,     Department of Internal Medicine A, Ernst-Moritz-Arndt-Universität Greifswald, Greifswald, Germany

Kara A. Mensink, MS, CGC,     Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, United States

Samuel C. Mok, PhD,     Department of Gynecologic Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, United States

Satdarshan P.S. Monga, MD,     Division of Experimental Pathology, Department of Pathology, Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, Pittsburgh Liver Research Center, University of Pittsburgh, School of Medicine, Pittsburgh, PA, United States

Thomas J. Montine, MD, PhD,     Department of Pathology, Stanford University, Palo Alto, CA, United States

Jason H. Moore, PhD,     Division of Informatics, Department of Biostatistics and Epidemiology, Institute for Biomedical Informatics, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States

Markus Morkel, PhD,     Laboratory of Molecular Tumor Pathology and Tumor Systems Biology, Charité – Universitätsmedizin Berlin, Berlin, Germany

Karl Munger, PhD,     Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA, United States

Zoltan Nagymanyoki, MD, PhD,     Department of Pathology, West Pacific Medical Laboratory, Santa Fe Springs, CA, United States

Robert D. Nerenz, PhD,     Assistant Professor of Pathology and Laboratory Medicine, Dartmouth-Hitchcock Medical Center, Lebanon, NH, United States

Alan L.-Y. Pang, PhD,     TGD Life Company Limited, Hong Kong Science Park, Shatin, Hong Kong SAR

Emanuel Petricoin, PhD,     Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, United States

Catherine Ptaschinski, PhD,     Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, United States

Reinhold Schäfer, PhD

Charité Comprehensive Cancer Center, Charité – Universitätsmedizin Berlin, Berlin, Germany

German Cancer Consortium (DKTK), German Cancer Research Center, Heidelberg, Germany

Matthias Sendler, MD,     Department of Internal Medicine A, Ernst-Moritz-Arndt-Universität Greifswald, Greifswald, Germany

Antonia R. Sepulveda, MD, PhD,     Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, NY, United States

Christine Sers, PhD,     Laboratory of Molecular Tumor Pathology and Tumor Systems Biology, Charité – Universitätsmedizin Berlin, Berlin, Germany

Lawrence M. Silverman, PhD,     Department of Pathology, University of Virginia Health System, Charlottesville, VA, United States

Joshua A. Sonnen, MD,     Department of Pathology, University of Utah, Salt Lake City, UT, United States

Christos Sotiriou, MD, PhD,     Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium

Roderick J. Tan, MD, PhD,     Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States

Gregory J. Tsongalis, PhD

The Audrey and Theodor Geisel School of Medicine at Dartmouth, Hanover, NH, United States

Laboratory for Clinical Genomics and Advanced Technology (CGAT), Department of Pathology and Laboratory Medicine, Dartmouth Hitchcock Medical Center, Lebanon, NH, United States

Vesarat Wessagowit, MD, PhD,     The Institute of Dermatology, Rajvithi Phyathai, Bangkok, Thailand

Eli S. Williams, PhD,     Department of Pathology, University of Virginia Health System, Charlottesville, VA, United States

Kwong-Kwok Wong, PhD,     Department of Gynecologic Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, United States

Dani S. Zander, MD,     Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH, United States

Dong-Er Zhang, PhD,     Department of Pathology and Division of Biological Sciences, University of California San Diego, La Jolla, CA, United States

Weidong Zhou, MD, PhD,     Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, United States

Preface

Pathology is the study of disease. The field of pathology emerged from the application of the scientific method to the study of human disease. Thus, pathology as a discipline represents the complementary intersection of medicine and basic science. Early pathologists were typically practicing physicians who described the various diseases that they treated and made observations related to factors that contributed to the development of these diseases. The description of disease evolved over time from gross observation to microscopic inspection of diseased tissues based on the light microscope and more recently to the ultrastructural analysis of disease with the advent of the electron microscope. As hospital-based and community-based registries of disease emerged, the ability of investigators to identify factors that cause disease and assign risk to specific types of exposures expanded to increase our knowledge of the epidemiology of disease. Although descriptive pathology can be dated to the earliest written histories of medicine and the modern practice of diagnostic pathology dates back perhaps 200  years, the elucidation of mechanisms of disease and linkage of disease pathogenesis to specific causative factors emerged more recently from studies in experimental pathology. The field of experimental pathology embodies the conceptual foundation of early pathology—the application of the scientific method to the study of disease—and applies modern investigational tools of cell and molecular biology to advanced animal model systems and studies of human subjects. The molecular era of biological science began over 60  years ago, whereas recent advances in our knowledge of molecular mechanisms of disease have propelled the field of molecular pathology. These advances were facilitated by significant improvements and new developments associated with the techniques and methodologies available to pose questions related to the molecular biology of normal and diseased states affecting cells, tissues, and organisms. Today, molecular pathology encompasses the investigation of the molecular mechanisms of disease and interfaces with translational medicine where new basic science discoveries form the basis for the development of new strategies for disease prevention, new therapeutic approaches and targeted therapies for the treatment of disease, and new diagnostic tools for disease diagnosis and prognostication.

With the remarkable pace of scientific discovery in the field of molecular pathology, basic scientists, clinical scientists, and physicians have a need for a source of information on the current state of the art of our understanding of the molecular basis of human disease. More importantly, the complete and effective training of today’s graduate students, medical students, postdoctoral fellows, and others, for careers related to the investigation and treatment of human disease, requires textbooks that have been designed to reflect our current knowledge of the molecular mechanisms of disease pathogenesis, as well as emerging concepts related to translational medicine. Most pathology textbooks provide information related to diseases and disease processes from the perspective of description (what does it look like and what are its characteristics), risk factors, disease-causing agents, and to some extent, cellular mechanisms. However, most of these textbooks lack in-depth coverage of the molecular mechanisms of disease. The reason for this is primarily historical—most major forms of disease have been known for a long time, but the molecular basis of these diseases is not always known or has been elucidated only very recently. However, with rapid progress over time and improved understanding of the molecular basis of human disease, the need emerged for new textbooks on the topic of molecular pathology, where molecular mechanisms represent the focus.

In this second edition of Molecular Pathology: The Molecular Basis of Human Disease, we have assembled a group of experts to discuss the molecular basis and mechanisms of major human diseases and disease processes, presented in the context of traditional pathology, with implications for translational molecular medicine. This volume is intended to serve as a multiuse textbook that would be appropriate as a classroom teaching tool for medical students, biomedical graduate students, allied health students, and others (such as advanced undergraduates). Furthermore, this textbook will be valuable for pathology residents and other postdoctoral fellows who desire to advance their understanding of molecular mechanisms of disease beyond what they learned in medical/graduate school. In addition, this textbook is useful as a reference book for practicing basic scientists and physician scientists who perform disease-related basic science and translational research, who require a ready information resource on the molecular basis of various human diseases and disease states. To be sure, our understanding of the many causes and molecular mechanisms that govern the development of human diseases is far from complete. Nevertheless, the amount of information related to these molecular mechanisms has increased tremendously in recent years, and areas of thematic and conceptual consensus have emerged. We have made an effort to integrate accepted principles with broader theoretical concepts in an attempt to present a current and comprehensive view of the molecular basis of human disease. We hope that this second edition of Molecular Pathology: The Molecular Basis of Human Disease will accomplish its purpose of providing students and researchers with in-depth coverage of the molecular basis of major human diseases in the context of traditional pathology so as to stimulate new research aimed at furthering our understanding of these molecular mechanisms of human disease and advancing the theory and practice of molecular medicine.

William B. Coleman

Gregory J. Tsongalis

Acknowledgments

The editors would like to acknowledge the significant contributions of a number of people to the successful production of the second edition of Molecular Pathology: The Molecular Basis of Human Disease.

We would like to thank the individuals who contributed to the content of this volume. The remarkable coverage of the state of the art in the molecular pathology of human disease would not have been possible without the hard work and diligent efforts of the 62 authors of the individual chapters. Many of these contributors are our long-time colleagues, collaborators, and friends, and they have contributed to other projects that we have directed, and we sincerely appreciate their willingness to contribute once again to a project that we found worthy. We especially thank the contributors to this volume who were willing to work with us for the first time. This group also includes some of our long-time friends and colleagues, as well as some new friends. We look forward to working with all of these authors again in the future. Each of these contributors provided us with an excellent treatment of their topic, and we hope that they will be proud of their individual contributions to the textbook. Furthermore, we would like to give a special thanks to our colleagues who coauthored chapters with us for this textbook. There is no substitute for an excellent coauthor when you are juggling the several responsibilities of concurrently editing and contributing to a textbook. Collectively, we can all be proud of this volume, as it is proof that the whole can be greater than the sum of its parts.

Thanks to Ms. Mara Conner (Senior Acquisitions Editor, Academic Press—Elsevier) who worked with us on the first edition of this textbook. She embraced the concept of this textbook when our ideas were not yet fully developed and encouraged us to pursue the project. She was receptive to the model for this textbook that we envisioned and worked closely with us to evolve the project into its final form. Without Mara’s early support, the first edition of this textbook would not have been so successful and this second edition would not have been possible.

We would also like to thank the many people who work for Academic Press—Elsevier that made this project possible. We have not met and do not know many of these people, but we appreciate their efforts to bring this textbook to its completed form. Special thanks goes to three key people who made significant contributions to this project on the publishing side and proved to be exceptionally competent and capable. Ms. Tari Broderick (Senior Acquisitions Editor, Academic Press—Elsevier) provided excellent oversight (and optimistic patience) during the construction and editing of this edition of the textbook and has become our valued colleague as we develop new projects. Ms. Lisa Eppich (Editorial Project Manager, Elsevier) provided excellent support to us throughout this project. As we interacted with our contributing authors, collected and edited manuscripts, and began production of the textbook, Lisa assisted us greatly by being a constant reminder of deadlines, helping us with communication with the contributors, and generally providing support for details small and large, all of which proved to be critical. Ms. Anusha Sambamoorthy (Project Manager, Elsevier) worked with us closely to ensure the integrity of the content of the textbook as it moved from the edited manuscripts into their final form. We thank her for her direct involvement with the production and also for directing her excellent production team. It was a pleasure to work with Tari, Lisa, and Anusha on this project. We hope that they enjoyed it as much as we did, and we look forward to working with them again soon.

William B. Coleman

Gregory J. Tsongalis

Chapter 1

Molecular Mechanisms of Cell Death

John J. Lemasters, MD, PhD     Departments of Drug Discovery & Pharmaceutical Sciences and Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, SC, United States

Abstract

Cell death is a fundamental pathophysiological process and also an essential event in normal life and development. Although many stimuli cause death of cells, the mode of cell death typically follows one of two patterns. The first is necrosis, or oncosis. Oncotic necrosis is most often the result of profound metabolic disruption and is characterized by cellular swelling leading to plasma membrane rupture with release of intracellular contents. The second pattern is apoptosis, a form of programmed cell death. Apoptosis causes the orderly resorption of individual cells initiated by well-defined pathways involving activation of proteases called caspases. In contrast to necrotic cell death, which typically occurs from adenosine triphosphate (ATP) depletion, apoptosis is an ATP-requiring process. Several distinct organelles (plasma membrane, mitochondrion, nucleus, endoplasmic reticulum, lysosome) give rise to signals that induce cell death. In particular, mitochondrial permeabilization and dysfunction typically develop in both necrosis and apoptosis. In some instances, apoptosis and necrosis share signaling pathways, as occurs in programmed necrosis called necroptosis. In this way, apoptosis and necrosis can represent extreme endpoints on a phenotypic continuum of lost cell viability.

Keywords

Apoptosis; Caspase; Death receptor; DISC; Mitochondrial permeability transition; Necroptosis; Necrosis; PARP; Reperfusion injury

Outline

Introduction

Modes of Cell Death

Structural Features of Necrosis and Apoptosis

Oncotic Necrosis

Apoptosis

Cellular and Molecular Mechanisms Underlying Necrotic Cell Death

Metastable State Preceding Necrotic Cell Death

Mitochondrial Dysfunction and ATP Depletion

Mitochondrial Uncoupling in Necrotic Cell Killing

Mitochondrial Permeability Transition

Inner Membrane Permeability

Mitochondrial Permeability Transition Pore

pH-dependent Ischemia/Reperfusion Injury

Role of the Mitochondrial Permeability Transition in pH-Dependent Reperfusion Injury

Oxidative Stress

Protein Kinase Signaling and the MPT

Other Stress Mechanisms Inducing Necrotic Cell Death

Poly (ADP-Ribose) Polymerase

Plasma Membrane Injury

Pathways to Apoptosis

Roles of Apoptosis in Biology

Plasma Membrane

Mitochondria

Cytochrome c Release

Regulation of the Mitochondrial Pathway to Apoptosis

Antiapoptotic Survival Pathways

Nucleus

Endoplasmic Reticulum

Lysosomes

Shared Pathways to Necrosis and Apoptosis

Programmed Necrosis

Ferroptosis

Pyroptosis

Necroptosis

Concluding Remark

Acknowledgments

References

Introduction

A common theme in disease is death of cells. In diseases ranging from stroke to congestive heart disease to alcoholic cirrhosis of the liver, death of individual cells leads to irreversible functional loss in whole organs and ultimately mortality. For such diseases, prevention of cell death becomes a basic therapeutic goal. By contrast in neoplasia, the purpose of chemotherapy is to kill proliferating cancer cells. For either therapeutic goal, understanding the mechanisms of cell death becomes paramount.

Modes of Cell Death

Although many stresses and stimuli cause cell death, the mode of cell death typically follows one of two patterns. The first is necrosis, a pathological term referring to areas of dead cells within a tissue or organ. Necrosis is typically the result of an acute and usually profound metabolic disruption, such as ischemia (loss of blood flow) and severe toxicant-induced damage. Since necrosis as observed in tissue sections is an outcome rather than a process, the term oncosis has been introduced to describe the process leading to necrotic cell death, but the term has yet to be widely adopted in the experimental literature [1–3]. Here, the terms oncosis, oncotic necrosis, and necrotic cell death will be used synonymously to refer both to the outcome of cell death and the pathogenic events precipitating cell killing.

The second pattern is programmed cell death, most commonly manifested as apoptosis, a term derived from an ancient Greek word for the falling of leaves in the autumn. In apoptosis, specific stimuli initiate execution of well-defined pathways leading to orderly resorption of individual cells with minimal leakage of cellular components into the extracellular space and little inflammation [4–6]. Whereas necrotic cell death occurs with abrupt onset after adenosine triphosphate (ATP) depletion, apoptosis may take hours to go to completion and is an ATP-requiring process without a clearly distinguished point of no return. Although apoptosis and necrosis were initially considered separate and independent phenomena, an alternate view is emerging that apoptosis and necrosis can share initiating factors and signaling pathways to become extremes on a phenotypic continuum variously called aponecrosis or necrapoptosis [7–9].

Structural Features of Necrosis and Apoptosis

Oncotic Necrosis

Cellular changes leading up to onset of necrotic cell death include formation of plasma membrane protrusions called blebs, mitochondrial swelling, dilatation of cisternae of the endoplasmic reticulum (ER) and nuclear membranes, dissociation of polysomes, and cellular swelling leading to rupture with release of intracellular contents (Table 1.1, Fig. 1.1). After necrotic cell death, characteristic histological features of loss of cellular architecture, vacuolization, karyolysis (dissolution of the nucleus), and increased eosinophilia soon become evident (Fig. 1.2). Cell lysis evokes an inflammatory response, attracting neutrophils and monocytes to the dead tissue to dispose of the necrotic debris by phagocytosis and defend against infection (Fig. 1.3). In organs like heart and brain with little regenerative capacity, healing occurs with scar formation, namely replacement of necrotic regions with fibroblasts and collagen, as well as other connective tissue components. In organs, such as the liver, that have robust regenerative capacity, cell proliferation can replace areas of necrosis with completely normal tissue within a few days. The healed liver tissue shows little or no residua of the necrotic event, but if regeneration fails, collagen deposition and fibrosis will occur instead to cause cirrhosis.

Apoptosis

Unlike necrosis, which often occurs in response to an imposed unphysiological stress, apoptosis is a process of physiological cell deletion that has an opposite role to mitosis in the regulation of cell populations. In apoptosis, cell death occurs with little release of intracellular contents, inflammation, and scar formation. Individual cells undergoing apoptosis separate from their neighbors and shrink rather than swell. Distinctive nuclear and cytoplasmic changes also occur, including chromatin condensation, nuclear lobulation and fragmentation, formation of numerous small cell surface blebs (zeiotic blebbing), and shedding of these blebs as apoptotic bodies that are phagocytosed by adjacent cells and macrophages for lysosomal degradation (Table 1.1, Fig. 1.3). Characteristic biochemical changes also occur, typically activation of a cascade of cysteine-aspartate proteases, called caspases, leakage of proapoptotic proteins like cytochrome c from mitochondria into the cytosol, internucleosomal deoxyribonucleic acid (DNA) degradation, degradation of poly(adenosine diphosphate [ADP] ribose) polymerase (PARP), and movement of phosphatidylserine to the exterior leaflet of the plasmalemmal lipid bilayer. Thus, apoptosis manifests a very different pattern of cell death than oncotic necrosis (Table 1.1, Fig. 1.3).

Table 1.1

Comparison of Necrosis and Apoptosis

Figure 1.1  Electron microscopy of oncotic necrosis to a rat hepatic sinusoidal endothelial cell after ischemia/reperfusion.

Note cell rounding, mitochondrial swelling (arrows), rarefaction of cytosol, dilatation of the ER and the space between the nuclear membranes (∗), chromatin condensation, and discontinuities in the plasma membrane. Bar is 2  μm.

Figure 1.2  Histology of necrosis after hepatic ischemia/reperfusion in a mouse.

Note increased eosinophilia, loss of cellular architecture, and nuclear pyknosis and karyolysis. Contrast to lower left and right areas that are nonnecrotic. Bar is 50  μm.

Figure 1.3  Scheme of necrosis and apoptosis.

In oncotic necrosis, swelling leads to bleb rupture and release of intracellular constituents, which attract macrophages that clear the necrotic debris by phagocytosis. In apoptosis, cells shrink and form small zeiotic blebs that are shed as membrane-bound apoptotic bodies. Apoptotic bodies are phagocytosed by macrophages and adjacent cells. Adapted with permission from Van CS, Van Den BW. Morphological and biochemical aspects of apoptosis, oncosis and necrosis. Anat Histol Embryol 2002;31:214–23.

Cellular and Molecular Mechanisms Underlying Necrotic Cell Death

Metastable State Preceding Necrotic Cell Death

Cellular events culminating in necrotic cell death are somewhat variable from one cell type to another, but certain events occur regularly. As implied by the term oncosis, cellular swelling is a prominent feature of oncotic necrosis [1,7]. In many cell types, swelling of 30%–50% occurs early after ATP depletion associated with formation of blebs on the cell surface (Fig. 1.4) [8,9]. These blebs contain cytosol and ER but exclude larger organelles like mitochondria and lysosomes. Bleb formation is likely due to cytoskeletal alterations after ATP depletion, whereas swelling arises from disruption of cellular ion transport [10,11]. Mitochondrial swelling and dilatation of cisternae of ER and nuclear membranes accompany bleb formation (Fig. 1.1). After longer times, a metastable state develops, which is characterized by mitochondrial depolarization, lysosomal breakdown, bidirectional leakiness of the plasma membrane to organic anions (but not cations), intracellular Ca²+ and pH dysregulation, and accelerated bleb formation with more rapid swelling [12–15]. The metastable state lasts only a few minutes and culminates in rupture of a plasma membrane bleb (Fig. 1.4) [13,14,16]. Bleb rupture leads to loss of metabolic intermediates such as those that reduce tetrazolium dyes, leakage of cytosolic enzymes like lactate dehydrogenase, uptake of dyes like trypan blue, and collapse of all electrical and ion gradients across the membrane. This all-or-nothing breakdown of the plasma membrane permeability barrier is long-lasting, irreversible, and incompatible with continued life of the cell.

Figure 1.4  Bleb rupture at onset of necrotic cell death.

After metabolic inhibition with cyanide and iodoacetate, inhibitors of respiration and glycolysis, respectively, a surface bleb of the cultured rat hepatocyte on the right has just burst. Note the discontinuity of the plasma membrane surface in the scanning electron micrograph. The hepatocyte on the left is also blebbed, but the plasma membrane is still intact, and viability has not yet been lost. Bar is 5  μm. Adapted with permission from Herman B, Nieminen AL, Gores GJ, Lemasters JJ. Irreversible injury in anoxic hepatocytes precipitated by an abrupt increase in plasma membrane permeability. FASEB J 1988;2:146–51.

Some work suggests that opening of a nonspecific anion channel in the plasma membrane initiates the metastable state [17,18]. Although potassium and sodium channels open early after metabolic disruption, cellular impermeability to chloride limits the rate of swelling. At onset of the metastable state, a relatively nonspecific chloride-conducting anion channel appears to open, permitting electroneutral uptake of electrolytes (principally sodium and chloride) and initiating rapid swelling driven by colloid osmotic (oncotic) forces (Fig. 1.5). Rapid swelling continues until one of the plasma membrane blebs ruptures. Bleb rupture is the final irreversible event precipitating cell death, since removal of the instigating stress (e.g., reoxygenation of anoxic cells) leads to cell recovery prior to bleb rupture but not afterwards [14]. Glycine and the glycine receptor antagonist strychnine protect against necrotic cell killing. Protection is associated with inhibition of this anion death channel and suppression of swelling in the metastable state. Glycine protection occurs without restoration of ATP or prevention of other metabolic derangements [11,18–21]. The glycine-gated chloride channel (GlyR) appears responsible for glycine cytoprotection, since glycine is not cytoprotective in cells not expressing the GlyRα1 subunit, and since GlyRα1 confers glycine cytoprotection in such cells [22]. Cytoprotection is mediated in part by the stress protein kinases, extracellular signal-regulated kinase-1 and -2 (ERK1/2), and serine/threonine-specific protein kinase B, also known as Akt [23].

Figure 1.5  Plasma membrane permeabilization leading to necrotic cell death.

Early after hypoxia and other metabolic stresses, ATP depletion leads to inhibition of the Na,K-ATPase and opening of monovalent cation channels causing cation gradients (Na+ and K+) to collapse. Swelling is limited by impermeability to anions. Later, glycine and strychnine-sensitive anion channels open to initiate anion entry and accelerate bleb formation and swelling. Swelling continues until a bleb ruptures. With abrupt and complete loss of the plasma membrane permeability barrier, viability is lost. Supravital dyes like trypan blue and propidium iodide enter the cell to stain the nucleus, and cytosolic enzymes like lactate dehydrogenase (LDH) leak out. With permission from Lemasters JJ, Qian T, He L, et al. Role of mitochondrial inner membrane permeabilization in necrotic cell death, apoptosis, and autophagy. Antioxid Redox Signal 2002;4:769–81.

Mitochondrial Dysfunction and ATP Depletion

Ischemia as occurs in strokes and heart attacks is perhaps the most common cause of necrotic cell killing. In ischemia, oxygen deprivation prevents ATP formation by mitochondrial oxidative phosphorylation, a process providing up to 95% of ATP utilized by highly aerobic tissues. The role of mitochondrial dysfunction in necrotic killing can be assessed experimentally by the ability of glycolytic substrates to rescue cells from lethal cell injury (Fig. 1.6) [24]. As an alternative source of ATP, glycolysis partially replaces ATP production lost after mitochondrial dysfunction. Maintenance of as little as 15% or 20% of normal ATP then rescues cells from necrotic death. Glucose and glycogen are prototypic glycolytic substrates that delay or prevent anoxic cell killing in most cell types. However, an important function of the liver is to maintain blood glucose levels constant, and hepatocytes do not consume glucose even during anoxia. For hepatocytes, fructose is a much better glycolytic substrate, and fructose but not glucose prevents loss of viability of hepatocytes during anoxia, respiratory inhibition, and inhibition of the mitochondrial ATP synthase [25,26].

Figure 1.6  Progression of mitochondrial injury.

Respiratory inhibition inhibits oxidative phosphorylation and leads to ATP depletion and necrotic cell death. Glycine blocks plasma membrane permeabilization causing necrotic cell death downstream of ATP depletion. Glycolysis restores ATP and prevents cell killing. Mitochondrial uncoupling as occurs after reperfusion due to the mitochondrial permeability transition (MPT) activates the mitochondrial ATPase to futilely hydrolyze glycolytic ATP, and protection against necrotic cell death is lost. By inhibiting the mitochondrial ATPase, oligomycin prevents ATP depletion and rescues cells from necrotic cell death if glycolytic substrate is present. With permission from Lemasters JJ, Qian T, He L, et al. Role of mitochondrial inner membrane permeabilization in necrotic cell death, apoptosis, and autophagy. Antioxid Redox Signal 2002;4:769–81.

In aerobic cells, exogenous glucose and fructose at high concentrations cause intracellular ATP to decrease because of ATP consumption by hexokinase and fructokinase, the first enzymes in the glycolytic metabolism of the respective two hexoses. As glucose-6-phosphate and other sugar phosphates accumulate, intracellular inorganic phosphate (Pi) also decreases [27,28]. In fructose-treated livers, decreased ATP has been interpreted as evidence of toxicity, but decreased Pi offsets the decline of ATP such that fructose-treated hepatocytes and livers maintain their ATP/ADP·Pi ratio (phosphorylation potential). Phosphorylation potential, rather than ATP concentration, ATP/ADP ratio or energy charge (defined as [ATP]  +  1/2 [ADP])/([ATP]  +  [ADP]  +  [AMP]), is the relevant thermodynamic variable reflecting cellular bioenergetic status [29]. Moreover, in anoxic livers and hepatocytes, glycolysis of fructose and endogenous glycogen increases ATP to protect against necrotic cell killing [25,26]. Fructose also protects against hepatocellular toxicity by oxidant chemicals, suggesting that mitochondria are also a primary target of cytotoxicity in oxidative stress [30].

Mitochondrial Uncoupling in Necrotic Cell Killing

Mitochondrial injury and dysfunction are progressive (Fig. 1.6). Anoxia and inhibition with a toxicant like cyanide inhibit respiration to cause ATP depletion and ultimately necrotic cell death. Glycolysis can replace this ATP supply, although only partially in highly aerobic cells, to rescue cells from necrotic killing. In the absence of respiration, mitochondrial membrane potential (ΔΨ) is sustained by reversal of the F1F0-ATPase synthase reaction. However, when mitochondria become permeable to protons (uncoupling), then maximal stimulation of F1F0-ATPase activity occurs. Since glycolytic ATP production cannot keep pace, ATP levels fall profoundly, mitochondria depolarize, and necrotic cell death ensues. In the presence of glycolytic substrate, oligomycin, an inhibitor of the F1F0-ATP synthase, prevents uncoupler-induced ATP depletion and subsequent cell death. Because oligomycin does not reverse uncoupling, mitochondrial ΔΨ is not restored [24,26]. Cytoprotection by oligomycin requires glycolytic ATP generation, since in the absence of glycolysis, oligomycin is itself toxic by inhibiting oxidative phosphorylation. Cytoprotection requiring the combination of glycolytic substrate and oligomycin indicates cytotoxicity mediated by mitochondrial uncoupling as shown, for example, for calcium ionophore toxicity and oxidative stress [30,31].

Mitochondrial Permeability Transition

Inner Membrane Permeability

In oxidative phosphorylation, respiration drives translocation of protons out of mitochondria to create an electrochemical proton gradient composed of a negative inside ΔΨ and an alkaline inside pH gradient (ΔpH). ATP synthesis is then linked to protons returning down this electrochemical gradient through the mitochondrial ATP synthase. This chemiosmotic proton circuit requires the mitochondrial inner membrane to be impermeable to ions and charged metabolites. Thus, metabolite exchange for oxidative phosphorylation occurs via specific transporters and exchangers in the inner membrane, including the adenine nucleotide translocator (ANT), which exchanges ATP for ADP; the phosphate transporter; and one of several transporter systems for uptake of respiratory substrates like pyruvate and fatty acids. By contrast, the outer membrane is nonspecifically permeable to ions and hydrophilic metabolites, which move across the outer membrane through a channel called the voltage-dependent anion channel (VDAC) [32,33]. Despite its name, VDAC has only weak anion selectivity and conducts freely most solutes up to a molecular mass of about 5  kDa.

Mitochondrial Permeability Transition Pore

In the mitochondrial permeability transition (MPT), the mitochondrial inner membrane abruptly becomes nonselectively permeable to solutes of molecular weight up to about 1500  Da [34,35]. Ca²+, oxidative stress, and numerous reactive chemicals induce the MPT, whereas cyclosporin A and pH less than 7 inhibit. Onset of the MPT causes mitochondrial depolarization, uncoupling, and large amplitude mitochondrial swelling driven by colloid osmotic forces. Opening of highly conductive permeability transition (PT) pores in the mitochondrial inner membrane underlies the MPT. Patch clamping shows that conductance through permeability transition pores (PT pores) is so great that opening of a single PT pore may be sufficient to cause mitochondrial depolarization and swelling [36].

The composition of PT pores is uncertain. In one model, PT pores are formed by ANT from the inner membrane, VDAC from the outer membrane, the cyclosporin A binding protein cyclophilin D (CypD) from the matrix, and possibly other proteins (Fig. 1.7A) [37,38]. Nonetheless, the MPT still occurs in mitochondria that are deficient in ANT and VDAC [39–41]. Moreover, although CypD is responsible for pore inhibition by cyclosporin A, a cyclosporin A-insensitive MPT still occurs in CypD-deficient mitochondria [42]. More recently, PT pores are proposed to form in association with the F1F0-ATP synthase, either in ATP synthase dimers (or higher order structures) at the interface between monomers or within the c-ring of the F0 portion (Fig. 1.7B); with spastic paraplegia 7 (SPG7), a mitochondrial AAA-type membrane protease, or with the inorganic phosphate carrier of the inner membrane [35,43–46]. An alternative model for the PT pore is that oxidative and other stresses damage membrane proteins that then misfold and aggregate to form PT pores in association with CypD and other molecular chaperones (Fig. 1.7C), which explains how multiple different membrane proteins can be nonexclusively involved in PT pore formation [47].

Figure 1.7  Models of mitochondrial permeability transition pores.

In one model (A), PT pores are composed of the ANT from the inner membrane (IM), cyclophilin D (CypD) from the matrix, and the voltage-dependent anion channel (VDAC) from the outer membrane (OM). Other proteins, such as the peripheral benzodiazepine receptor (PBR), hexokinase (HK), creatine kinase (CK), and Bax may also contribute. PT pore openers include Ca²+, inorganic phosphate (Pi), reactive oxygen and nitrogen species (ROS, RNS), and oxidized pyridine nucleotides (NAD(P)+) and glutathione (GSSG). A newer model (B) has PT pores forming in F1F0-ATP synthase dimers at the interface between monomers (or possibly in association with c-rings). OSCP (oligomycin sensitivity-conferring protein), a, b, c, d, e, f, g, α, β, γ, δ, ε, A6L, and F8 are subunits of the synthase. An alternative proposal (C) suggests that oxidative and other damage to integral inner membrane proteins leads to misfolding. These misfolded proteins aggregate at hydrophilic surfaces facing the hydrophobic bilayer to form aqueous channels. CypD and other chaperones block conductance of solutes through these nascent PT pores. High-matrix Ca²+ acting through CypD leads to PT pore opening, an effect blocked by cyclosporin A (CsA). As misfolded protein clusters exceed the number of chaperones to regulate them, constitutively open channels form. Such unregulated PT pores are not dependent on Ca²+ for opening and are not inhibited by CsA. Adapted with permission from Kim JS, He L, Qian T, Lemasters JJ. Role of the mitochondrial permeability transition in apoptotic and necrotic death after ischemia/reperfusion injury to hepatocytes. Curr Mol Med 2003;3:527–35.

pH-dependent Ischemia/Reperfusion Injury

Ischemia is an interruption of blood flow and hence oxygen supply to a tissue or organ. In ischemic tissue, anaerobic glycolysis, hydrolysis of ATP, and release of protons from acidic organelles cause tissue pH to decrease by a unit or more. The naturally occurring acidosis of ischemia actually protects against onset of necrotic cell death. Acidosis also dramatically delays cell killing from oxidant chemicals, ionophores, and alkylating agents [12,48–50].

Although acidosis protects against cell killing during ischemia, reoxygenation and recovery of pH after reperfusion act to precipitate necrotic cell death. In cultured cells and perfused organs, ischemia/reperfusion injury can be reproduced using anoxia at acidotic pH to simulate ischemia followed by reoxygenation at normal pH to simulate reperfusion. Reperfusion in this model causes necrotic cell killing with release of intracellular enzymes like lactate dehydrogenase and nuclear labeling with vital dyes like trypan blue and propidium iodide [11,12,51–53]. Much of reperfusion injury leading to necrotic cell death is attributable to recovery of pH, since reoxygenation at low pH prevents cell killing entirely, whereas restoration of normal pH without reoxygenation produces similar cell killing as restoration of pH with reoxygenation, a so-called pH paradox (Fig. 1.8).

Figure 1.8  Mitochondrial inner membrane permeabilization in adult rat cardiac myocytes after ischemia and reperfusion.

After loading mitochondria of cardiac myocytes with calcein, cells were subjected to 3  h of anoxia at pH 6.2 (ischemia) followed by reoxygenation at pH 7.4 (A), pH 6.2 (B), or pH 7.4 with 1  μM CsA (C). Red-fluorescing propidium iodide was present to detect loss of cell viability. Note that green calcein fluorescence was retained by mitochondria at the end of ischemia (1  min before reperfusion), indicating that PT pores had not opened. After reperfusion at pH 7.4, mitochondria progressively released calcein over 30  min, at which time calcein was nearly evenly distributed throughout cytosol. After 60  min, all cellular calcein was lost, and the nucleus stained with PI, indicating loss of viability. After reperfusion at pH 6.2 (B) or at pH 7.4 in the presence of CsA (C), calcein was retained and cell death did not occur. Thus, reperfusion at pH 7.4 induced onset of the MPT and necrotic cell death that were blocked with CsA and acidotic pH. Adapted with permission from Kim JS, Jin Y, Lemasters JJ. Reactive oxygen species, but not Ca²+ overloading, trigger pH- and mitochondrial permeability transition-dependent death of adult rat myocytes after ischemia-reperfusion. Am J Physiol Heart Circ Physiol 2006;290:H2024–34.

Cell death in the pH paradox is linked to intracellular pH. Ionophores like monensin that accelerate recovery of intracellular pH from the acidosis of ischemia after reperfusion also accelerate necrotic cell killing [51]. Conversely, inhibition of Na+/H+ exchange with dimethylamiloride or Na+-free medium delays recovery of intracellular pH and prevents reperfusion-induced necrotic cell killing almost completely [51,52,54,55]. Cell killing in the pH paradox is linked specifically to intracellular pH and occurs independently of changes of cytosolic and extracellular free Na+ and Ca²+ [12,51,54–57].

Role of the Mitochondrial Permeability Transition in pH-Dependent Reperfusion Injury

pH below 7 inhibits PT pores, and recovery of intracellular pH to 7 or greater after reperfusion induces the MPT, as shown directly by confocal/multiphoton microscopy [52,53]. During ischemia, mitochondria depolarize because of respiratory inhibition, but the mitochondrial inner membrane remains impermeant to fluorophores like calcein that can only pass through PT pores (Fig. 1.8). After reperfusion at normal pH, mitochondria repolarize initially, as shown by uptake of membrane potential–indicating fluorophores (Fig. 1.9). Subsequently and in parallel with recovery of intracellular pH to neutrality, the MPT occurs, leading to permeabilization of the inner membrane to calcein and mitochondrial depolarization (Figs. 1.8 and 1.9). ATP depletion then follows, and necrotic cell death occurs.

Reperfusion at acidotic pH to prevent recovery of pH and reperfusion in the presence of PT pore blockers (e.g., cyclosporin A and its derivatives) prevents mitochondrial inner membrane permeabilization, depolarization, and cell killing (Figs. 1.8 and 1.9). Notably, cyclosporin A protects when added only during the reperfusion phase, as now confirmed by decreased infarct size in patients receiving percutaneous coronary intervention for ischemic heart disease [46,52,53,58,59], but see [60]. Thus, the MPT is the proximate cause of pH-dependent cell killing in ischemia/reperfusion injury.

Figure 1.9  Mitochondrial ROS formation after reperfusion.

Myocytes were coloaded with red-fluorescing tetramethylrhodamine methyester (TMRM) and green-fluorescing chloromethyldichlorofluorescin (cmDCF) to monitor mitochondrial membrane potential and ROS formation, respectively. At the end of 3  h of ischemia, mitochondria were depolarized (lack of red TMRM fluorescence). After 20  min of reperfusion, mitochondria took up TMRM, indicating repolarization, and cmDCF fluorescence increased progressively inside mitochondria (A). Subsequently, hypercontraction and depolarization occurred after 40  min, and viability was lost within 120  min, as indicated by nuclear labeling with red-fluorescing propidium iodide. When cyclosporin A was added at reperfusion (B), mitochondria underwent sustained repolarization, and hypercontracture and cell death did not occur. Nonetheless, mitochondrial cmDCF fluorescence still increased. By contrast, reperfusion with antioxidants prevented ROS generation and MPT onset with subsequent cell death (data not shown). Thus, mitochondrial ROS generation induces the MPT and cell death after ischemia/reperfusion. Adapted with permission from Kim JS, Jin Y, Lemasters JJ. Reactive oxygen species, but not Ca²+ overloading, trigger pH- and mitochondrial permeability transition-dependent death of adult rat myocytes after ischemia-reperfusion. Am J Physiol Heart Circ Physiol 2006;290:H2024–34.

Minocycline, a semisynthetic tetracycline derivative that protects against neurodegenerative disease, trauma, hepatoxicity, hemorrhagic shock/resuscitation, and hypoxia–ischemia [61–64] also inhibits the MPT and protects against cell killing after reperfusion of livers stored by cold ischemia for transplantation [65]. However, the mechanism of MPT blockade differs from cyclosporin A. Rather than blocking through an interaction with CypD, minocycline blocks the MPT by inhibiting mitochondrial calcium uptake. This observation implies that calcium uptake (or uptake of other divalent cation by the same pathway) into mitochondria is a prerequisite for MPT onset after reperfusion.

Oxidative Stress

Reactive oxygen species (ROS) and reactive nitrogen species (RNS), including superoxide, hydrogen peroxide, hydroxyl radical, and peroxynitrite, have long been implicated in cell injury leading to necrosis (Fig. 1.10). In hepatocytes, mitochondrial NAD(P)H oxidation after oxidative stress disrupts mitochondrial Ca²+ homeostasis to cause an increase of mitochondrial Ca²+, which in turn stimulates intramitochondrial ROS formation and onset of the MPT [15,30,66]. In cardiac myocytes after reperfusion, intramitochondrial ROS formation also occurs to initiate the MPT and subsequent necrotic cell death (Fig. 1.9) [53]. Notably, inhibition of the MPT with cyclosporin A does not prevent mitochondrial ROS generation after reperfusion (Fig. 1.9). Although intramitochondrial Ca²+ may be permissive for MPT onset after reperfusion, massive Ca²+ overloading and hypercontracture in myocytes does not occur until after the MPT, and MPT inhibitors prevent Ca²+ overloading and cell death [53]. In neurons, excitotoxic stress with glutamate and N-methyl-D-aspartate receptor agonists also stimulates mitochondrial ROS formation, leading to the MPT and excitotoxic injury [67–70].

Iron potentiates injury in a variety of diseases and is an important catalyst for hydroxyl radical formation from superoxide and hydrogen peroxide (Fig. 1.10) [71–74]. Increased intracellular chelatable iron contributes to cell death after cold ischemia/reperfusion, and addition of a membrane permeable Fe³+ complex causes the MPT and consequent necrotic and apoptotic cell death [75–77].

During oxidative stress, acetaminophen hepatotoxicity, and hypoxia/ischemia, lysosomes rupture, which releases chelatable (loosely bound) iron with consequent prooxidant cell damage [78–83]. This iron is taken up into mitochondria by the mitochondrial calcium uniporter and helps catalyze mitochondrial ROS generation. Iron chelation with desferal prevents mitochondrial ROS formation and decreases cell death [53,62,82,84].

Figure 1.10  Iron-catalyzed free radical generation.

Oxidative stress causes oxidation of GSH and NAD(P)H, important reductants in antioxidant defenses, promoting increased net formation of superoxide (O2−) and hydrogen peroxide (H2O2). Superoxide dismutase converts superoxide to hydrogen peroxide, which is further detoxified to water by catalase and peroxidases. In the iron-catalyzed Haber Weiss reaction (or Fenton reaction), superoxide reduces ferric iron (Fe³+) to ferrous iron (Fe²+), which reacts with hydrogen peroxide to form the highly reactive hydroxyl radical (OH•). Hydroxyl radical reacts with lipids to form alkyl radicals (L) that initiate an oxygen-dependent chain reaction generating peroxyl radicals (LOO•) and lipid peroxides (LOOH). Iron also catalyzes a chain reaction generating alkoxyl radicals (LO•) and more peroxyl radicals. Nitric oxide synthase catalyzes formation of nitric oxide (NO) from arginine. Nitric oxide reacts rapidly with superoxide to form unstable peroxynitrite anion (ONOO−), which decomposes to nitrogen dioxide and hydroxyl radical. In addition to attacking lipids, these radicals also attack proteins and nucleic acids.

Protein Kinase Signaling and the MPT

Reperfusion with nitric oxide suppresses MPT onset and reperfusion-induced cell killing after ischemia [85]. A signaling cascade of guanylyl cyclase, cyclic guanosine monophosphate (cGMP), and cGMP-dependent protein kinase (protein kinase G) mediates nitric oxide protection against MPT onset. In isolated mitochondria, a combination of cGMP, cytosolic extract as a source of protein kinase, and ATP blocks the Ca²+-induced MPT. Thus, protein kinases act directly on mitochondria to negatively regulate the MPT [85,86]. Protein kinase C epsilon (PKCε), together with inhibition of glycogen synthase kinase-3 beta (GSK-3β), also leads to MPT inhibition, whereas c-Jun nuclear kinase-2 (JNK-2) promotes the MPT in reperfusion injury and acetaminophen hepatotoxicity [87–93].

Other Stress Mechanisms Inducing Necrotic Cell Death

Poly (ADP-Ribose) Polymerase

Single-strand breaks induced by ultraviolet light, ionizing radiation, and ROS (particularly hydroxyl radical and peroxynitrite) activate PARP isoforms 1 and 2 (PARP-1/2). PARP assists in the repair of single-strand DNA breaks by recruiting scaffolding proteins, DNA ligases, and polymerases that mediate base-excision repair. With excess DNA damage, PARP attaches ADP-ribose to the strand breaks and elongates ADP-ribose polymers attached to the DNA. Consumption of the oxidized form of nicotinamide adenine dinucleotide (NAD+) in this fashion leads to NAD+ depletion, disruption of ATP-generation by glycolysis and oxidative phosphorylation, and ATP depletion-dependent cell death [94,95]. Mice deficient in PARP-1 or PARP-2 and mice treated with PARP inhibitors are protected against such DNA damage–induced necrosis [96]. Glycosidases cleave long ADP-ribose polymers attached to DNA into large oligomers. Such oligomers can translocate to mitochondria to cause mitochondrial dysfunction and possibly the MPT [97]. Without excess DNA damage, PARP promotes repair of DNA single strand breaks via the base-excision repair/single-strand break repair pathway. Thus by interfering with this DNA