M E T H O D S I N M O L E C U L A R M E D I C I N ETM Atherosclerosis Experimental Methods and Protocols Edited by Angela F. Drew Humana Press Atherosclerosis M E T H O D S I N M O L E C U L A R M E D I C I N ETM John M. Walker, SERIES EDITOR 60. Interleukin Protocols, edited by Luke 47. Vision Research Protocols,edited by A. J. O’Neill and Andrew Bowie, 2001 P. Elizabeth Rakoczy, 2001 59. Molecular Pathology of the Prions, 46. Angiogenesis: Reviews and edited by Harry F. Baker, 2001 Protocols,edited byJ. Clifford 58. Metastasis Research Protocols: Murray, 2001 Volume 2, Cell Behavior In Vitro 45. Hepatocellular Carcinoma: Methods and In Vivo, edited by Susan A. and Protocols, edited by Nagy A. Brooks and Udo Schumacher, 2001 Habib, 2000 57. Metastasis Research Protocols: 44. Asthma: Mechanisms and Protocols, Volume 1, Analysis of Cells and edited by K. Fan Chung and Ian Tissues,edited by Susan A. Brooks Adcock, 2001 andUdo Schumacher, 2001 43. Muscular Dystrophy: Methods and 56. Human Airway Inflammation: Protocols,edited by Katherine B. Sampling Techniques and Analytical Bushby and Louise Anderson, 2001 Protocols,edited by Duncan F. Rogers 42. Vaccine Adjuvants: Preparation and Louise E. Donnelly, 2001 Methods and Research Protocols, 55. Hematologic Malignancies: Methods edited by Derek T. O’Hagan, 2000 and Protocols, edited by Guy B. Faguet, 41. Celiac Disease: Methods and 2001 Protocols, edited by Michael N. 54. Mycobacterium Tuberculosis Marsh,2000 Protocols,edited by Tanya Parish and Neil G. Stoker, 2001 40. Diagnostic and Therapeutic Antibodies,edited by Andrew J. T. 53. Renal Cancer: Methods and Protocols, George and Catherine E. Urch, 2000 edited by Jack H. Mydlo, 2001 39. Ovarian Cancer: Methods and Protocols, 52. Atherosclerosis: ExperimentalMethods edited by John M. S. Bartlett, 2000 and Protocols,edited by Angela F. Drew, 2001 38. Aging Methods and Protocols, 51. Angiotensin Protocols, edited by edited by Yvonne A. Barnett and Donna H. Wang, 2001 Christopher R. Barnett, 2000 50. Colorectal Cancer: Methods and 37. Electrochemotherapy, Protocols, edited by Steven M. Electrogenetherapy, and Transdermal Powell,2001 Drug Delivery: Electrically Mediated Delivery of Molecules to Cells, edited by 49. Molecular Pathology Protocols, Mark J. Jaroszeski, Richard Heller, and edited by Anthony A. Killeen, 2001 Richard Gilbert, 2000 48. Antibiotic Resistance Methods and 36. Septic Shock Methods and Protocols, Protocols,edited by Stephen H. edited by Thomas J. Evans, 2000 Gillespie,2001 M E T H O D S I N M O L E C U L A R M ED I C I N ETM Atherosclerosis Experimental Methods and Protocols Edited by Angela F. Drew Children's Hospital Research Foundation Cincinnati, OH Humana Press Totowa, New Jersey © 2001 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. 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Cover art: The cover image shows oil red O stained arteries from typical 20–22-wk-old apoliprotein E-deficient and plasminogen-deficient mice. (Top left) Plasminogen-deficient mice have no obvious lipid deposits around the valve cusp. (Top right) Apolipoprotein E-deficient mice develop sponta- neous lipid-containing fatty streak lesions. Mice with simultaneous plasminogen- and apolipoprotein E-deficiencies develop larger fatty streak lesions (bottom left) and more advanced fibroproliferative plaques with organized smooth muscle cell caps and medial disruption (bottom right). This illustra- tion was kindly provided by Dr. Qing Xiao. Cover design by Patricia F. Cleary. For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel: 973-256-1699; Fax: 973-256-8341; E-mail: [email protected], or visit our Website at www.humanapress.com. 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II. series. [DNLM: 1. Atherosclerosis. 2. Research--methods. WG 550 A86957 2001 RC692.A7288 2001 616.19369972--dc21 00-038924 Preface Atherosclerosis: Experimental Methods and Protocols aims to provide the reader with a compilation of techniques that will prove useful to active investigators across the field of experimental atherosclerosis research. In fact, this volume is unique, the first devoted to a broad spectrum of techniques and assays, some adopted from other disciplines, not previously brought together in one book. Our approach is designed to permit researchers to select the techniques that will answer their particular sets of questions, in any of the expanding number of both animal models and in vitro systems now available for studying factors contributing to the development or progression of athero- sclerotic lesions. Researchers can only benefit from this collection of relevant techniques, written and explained by experts in each of these fields. Both investigators beginning in the field of atherosclerosis studies and researchers entering the field from related but different areas of study will benefit from Atherosclerosis: Experimental Methods and Protocols. Sufficient background is provided for a beginner to carry out the techniques described in the chapters, yet great depth is achieved owing to the special expertise of the authors. Researchers new to the field of atherosclerosis will appreciate the benefits of having these techniques gathered in one volume for their investi- gations. In addition, researchers already in the field of atherosclerosis research may benefit from the wide array of techniques and ideas provided by enjoying expanded opportunities to investigate their hypotheses. Practical information regarding sample collection, choice of model system, experimental design, and data analysis techniques are each provided in these chapters. In addition to methods for both well-documented and novel tech- niques, chapters summarizing general aspects of atherosclerosis research, such as animal models, are included. A summary of newly emerging animal models, in particular, genetically manipulated mice, provides sufficient information to become involved in this exciting new area of research. Assay systems for serum or plasma determination are becoming increasingly relevant in diagnostic and epidemiological studies, and have accordingly been described in many of the chapters. Both traditional and newer methods for identifying and separat- ing classes and subclasses of lipoproteins have been included, along with v vi Preface assays for more recently identified plasma proteins implicated in atherogen- esis, such as cholesteryl ester transfer protein, homocysteine, glycated lipo- proteins, and apolipoprotein(a). Chapters describing the isolation and culture of cells and glycosaminoglycans from atherosclerotic plaques follow, and may form the basis of many in vitro assays. In vivo techniques for the collection and analysis of experimental atherosclerotic lesions have been included in the later chapters. Finally, a comprehensive overview describing gene therapies under recent investigation in the field of atherosclerosis/restenosis—along with a practical example of successful implementation of such a therapy in pigs— have been included. Obtaining a general overview of the material included in Atherosclerosis: Experimental Methods and Protocols, prior to a more detailed study of particular chapters, will illuminate many facts broadly related to atheroscle- rosis that may be useful initially, or later, in the course of a research program. Included in the “Notes” section of each chapter is additional information sufficient for successful application of the technique. Often this kind of detail is lacking from brief methodological descriptions in the literature. From their excellent working knowledge of the techniques described, our authors may save a new researcher much time and effort. The compil- ation of these techniques into a single volume will hopefully benefit many researchers in pursuit of understanding, diagnosing, and ultimately preventing or treating atherosclerosis. Angela F. Drew Contents Preface ............................................................................................................v Contributors....................................................................................................ix 1 Animal Models of Diet-Induced Atherosclerosis Angela F. Drew.......................................................................................1 2 Mechanical Injury Models: Balloon Catheter Injury to Rat Common Carotid Artery Rodney J. Dilley.....................................................................................7 3 Genetically Manipulated Models of Atherosclerosis in Mice Qing Xiao..............................................................................................15 4 Lipoprotein Isolation and Analysis from Serum by Preparative Ultracentrifugation Kishor M. Wasan, Shawn M. Cassidy, Allison L. Kennedy, and Kathy D. Peteherych............................27 5 Separation of Plasma Lipoproteins in Self-Generated Gradients of Iodixanol Joan A. Higgins, John M. Graham, and Ian G. Davies....................37 6 Fractionation of Lipoprotein Subclasses in Self-Generated Gradients of Iodixanol John M. Graham, Bruce A. Griffin, Ian G. Davies, and Joan A. Higgins........................................................................51 7 Detecting Cholesteryl Ester Transfer Protein in Plasma Andreas Ritsch and Josef R. Patsch................................................61 8 Determination of Plasma Homocysteine Roberto Accinni and Oberdan Parodi...............................................77 9 Assay for Serum Glycated Lipoproteins Akira Tanaka.......................................................................................105 10 Lipoprotein(a) Quantitation Michael Bender and Werner Gross.................................................113 11 Isolation and Characterization of Glycosaminoglycans from Human Atheromatous Vessels Eleni Papakonstantinou, Michael Roth, and George Karakiulakis..............................................................123 vii viii Contents 12 Culture of Human Smooth Muscle Cells Marisa A. Gallicchio..........................................................................137 13 Culture of Human Endothelial Cells Marisa A. Gallicchio..........................................................................147 14 Assay for Cell Migration and Invasion of Vascular Smooth Muscle Cells George Karakiulakis, Eleni Papakonstantinou, and Michael Roth...........................................................................163 15 Collection and Processing of Arterial Specimens for Histological Analysis Angela F. Drew...................................................................................171 16 Intravascular Ultrasound Imaging in the Quantitation of Atherosclerosis in Vivo Hannu I. Manninen.............................................................................177 17 Identification of Cell Types and Quantification of Lesion Composition Rodney J. Dilley.................................................................................187 18 Nonradioactive In Situ Hybridization in Atherosclerotic Tissue Jim Apostolopoulos..........................................................................195 19 Evaluation of Angiogenesis Marcy Silver and Jeffrey M. Isner....................................................207 20 Evaluation of Cell Proliferation in Human Atherosclerotic Lesions Elena R. Andreeva and Alexander N. Orekhov..............................213 21 Gene Transfer to the Vasculature: Historical Perspective and Implications for Future Research Objectives Sarah J. George and Andrew H. Baker...........................................219 22 A Pig Model of Vein Graft Disease: Applications for Potential Gene Therapies Clinton T. Lloyd, Sarah J. George, Gianni D. Angelini, Andrew C. Newby, and Andrew H. Baker...................................233 Index............................................................................................................245 Contributors ROBERTO ACCINNI • Clinical Physiology Institute of CNR, Section of Milan, Department of Cardiology A. De Gasperis, Ospedale Niguarda Cà Granda, Milan, Italy ELENA R. ANDREEVA • Institute of Experimental Cardiology, Cardiology Research Center, and Institute for Atherosclerosis Research, Russian Academy of Natural Sciences, Moscow, Russia GIANNI D. ANGELINI • Department of Medicine and Therapeutics, University of Glasgow, Glasgow, United Kingdom JIM APOSTOLOPOULOS• Research Department, Australian Red Cross Blood Service, Victoria, Australia ANDREW H. BAKER• Department of Medicine and Therapeutics, University of Glasgow, Glasgow, United Kingdom MICHAEL BENDER • Gustav Embden Centre of Biological Chemistry, Johann Wolfgang Goethe University, Frankfurt-am-Main, Germany SHAWN M. CASSIDY • Division of Pharmaceutics and Biopharmaceutics, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada IAN G. DAVIES • School of Biomolecular Sciences, Liverpool John Moores University, Liverpool, United Kingdom RODNEY J. DILLEY • Morphology Laboratory, Baker Medical Research Institute, Melbourne, Victoria, Australia ANGELA F. DREW • Division of Developmental Biology, Children's Hospital Research Foundation, Cincinnati, OH MARISA A. GALLICCHIO • Department of Medicine, Austin and Repatriation Medical Centre, University of Melbourne, Heidelberg, Victoria, Australia SARAH J. GEORGE• Department of Medicine and Therapeutics, University of Glasgow, Glasgow, United Kingdom JOHN M. GRAHAM • School of Biomolecular Sciences, Liverpool John Moores University, Liverpool, United Kingdom BRUCE A. GRIFFIN • Centre for Nutrition and Food Safety, Department of Biological Sciences, University of Surrey, Guildford, United Kingdom ix