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Atherosclerotic plaque ultrastructure following exposure of Watanabe heritable hyperlipidemic rabbits… Tranfield, Erin M. 2007

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ATHEROSCLEROTIC PLAQUE ULTRASTRUCTURE FOLLOWING EXPOSURE OF WATANABE HERITABLE HYPERLIPIDEMIC RABBITS TO PARTICULATE MATTER AIR POLLUTION by E R I N M . T R A N F I E L D B . S c , University of Victoria, 2002 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Pathology) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A M a y 2007 © Erin M . Tranfield, 2007 Abstract Epidemiological studies indicate that acute cardiovascular events follow exposure to particulate matter air pollution (PMio) and preliminary animal studies have associated repeated PMio exposure with progressive changes in atherosclerotic plaques. The unexplored questions are those addressing the precise physiological and pathological cellular mechanisms that may account for the positive association between cardiopulmonary morbidity and mortality and PMio exposure. The overarching objective of these studies was to examine atherosclerotic plaques from rabbits exposed to PMio for morphological signs of remodelling and reorganization, with particular emphasis placed on the integrity o f the endothelium and its underlying extracellular matrix; the cell population subtending the endothelium; and evidence of cap remodelling and destabilization. Watanabe heritable hyperlipidemic rabbits were exposed to 5 mg of urban PMio (EHC-93) (n = 8) or saline alone (n = 8) by instillation down the upper airways twice per week for four weeks. Following sacrifice, the abdominal aorta was processed for light and electron microscopy. Light microscopy and transmission electron microscopy qualitative and quantitative analyses were done on tissues processed for transmission electron microscopy. Samples were also processed for and analyzed using scanning electron microscopy. Light microscopy imaging revealed a significant accumulation o f macrophage-derived foam cells immediately below the plaque endothelium of W H H L rabbits repeatedly exposed to PMio (p = 0.04). Qualitative electron microscopy observations showed an organizational change to the atherosclerotic plaques o f P M i 0 exposed W H H L rabbits evidenced by reduced plaque stratification, an absence of a distinct necrotic core and increased macrophage-derived foam cells subtending the endothelium. Using both 2- and 3-dimensional analyses, electron microscopy investigations demonstrated that the insinuation o f macrophage-derived foam n cells immediately below the endothelium o f atherosclerotic caps of PMio exposed rabbits was associated with the separation of the endothelium from the reticulum of dense E C M (p < 0.0001), fragmentation of the reticulum of dense E C M and a significant increase in endothelial contact with macrophage-derived foam cells (p < 0.0039). Scanning electron microscopy analysis of plaques from control and PMio treated W H H L rabbits revealed intense leukocyte adhesion and emigration over the plaque core of the PMio exposed rabbits. A s expected, leukocyte adhesion and transmigration were observed at the shoulder regions of atherosclerotic plaques from both control rabbits and PMio exposed rabbits and the surface morphology of the endothelium differed between the normal blood vessel wall and areas of atherosclerotic disease. PMio exposure promotes atherosclerotic plaque destabilization by the migration of macrophage-derived foam cells from the core of the atherosclerotic plaque, to the atherosclerotic cap regions and even into the systemic circulation. In the process o f the migration, the reticulum of dense E C M subtending the endothelium is degraded compromising the stability of the endothelium over the atherosclerotic plaque cap. i i i Table of Contents Abstract ii Table of Contents iv List of Tables vii List of Figures viii Abbreviations xi Acknowledgements xiii Dedication xv 1 Introduction 1 1.1 The History o f A i r Pollution 1 1.2 The Causative Agent: Particulate Matter A i r Pollution 3 1.2.1 Particle Size 4 1.2.2 Particle Composition 7 1.2.3 Particle Shape 10 1.2.4 Particle Dispersion in the Lungs 10 1.2.5 Existence of a Threshold , 11 1.2.6 Lag Period : 12 1.3 Morbidity and Mortality associated with PMio Exposure 12 1.4 Atherosclerosis 14 1.4.1 Global Burden of Disease 14 1.4.2 Risk Factors for Atherosclerosis 16 1.4.3 A Brief History of Our Understanding of Atherosclerosis 16 1.4.4 Pathogenesis of Atherosclerosis: A Chronic Inflammatory Disease 18 1.4.5 Shear Stress and Atherosclerotic Plaque Development and Progression 19 1.4.6 Pathological Classification of Atherosclerosis 19 1.4.7 The Danger o f Atherosclerotic Plaques 25 1.4.8 Stable versus Unstable Atherosclerotic Plaques 27 1.5 Proposed Mechanisms for Particulate Matter Mediated Cardiovascular Events 28 1.5.1 Inflammatory Mechanism 29 1.5.2 Dysfunction of the Autonomic Nervous System 31 1.5.3 Cardiac Malfunction 32 1.6 Hypothesis 33 1.6.1 Specific Aims 34 2 General Methodology 35 2.1 Animals 35 2.2 Urban Particulate Matter 36 2.3 PMio Exposure 3 9 iv 2.3.1 Exposure Level 39 2.3.2 Exposure Protocol 39 2.4 Tissue Fixation, Excision and Processing for Electron Microscopy 40 2.4.1 Inclusion Criteria for Tissue Analysis 42 2.5 Tissue Fixation, Excision and Processing for Histochemistry and Immunohistochemistry 43 3 Alterations in Atherosclerotic Plaque Organization 44 3.1 Introduction 44 3.2 A i m 47 3.3 Methodology 47 3.3.1 Qualitative Protocol 47 3.3.2 Quantitative Protocol 47 3.4 Results 48 3.4.1 The Basis of the 23 um Depth o f Interest 48 3.4.2 Qualitative Observations 49 3.4.3 Quantitative Results 53 3.5 Discussion 53 4 Endothelial Cell Contacts 56 4.1 Introduction 56 4.2 Aims 58 4.3 Methodology 58 4.3.1 Qualitative Observations 58 4.3.2 Morphometric Analysis 58 4.3.3 3 -Dimensional Reconstruction 64 4.4 Results 64 4.4.1 Qualitative Observations 64 4.4.2 Morphometric Analysis : 80 4.4.3 3-Dimensional Reconstruction 83 4.5 Discussion 88 5 Composition of Endothelial Basal Lamina 94 5.1 Introduction 94 5.2 A i m 96 5.3 Methodology 96 5.3.1 Microanatomical Staining 96 5.3.2 Immunohistochemical Staining 98 5.4 Results 99 5.4.1 Microanatomical Staining 99 5.4.2 Immunohistochemical Staining 100 5.5 Discussion 107 6 Surface Morphology of Atherosclerotic Plaques 110 6.1 Introduction 110 6.2 A i m 112 6.3 Methodology 112 6.4 Results 112 6.5 Discussion 127 7 Summary and Discussion , 131 7.1 Restatement of the Problem 131 7.2 Summary of Findings 132 7.3 Findings in the Context of the Scientific Literature 133 7.3.1 Macrophage-Derived Foam Cel l Accumulation 133 7.3.2 Separation of the Endothelial Cells from the E C M and Its Degradation 134 7.3.3 Reduction of Acid ic and Sulphated Proteoglycan Moieties 135 7.3.4 Macrophage-Derived Foam Cel l Emigration 136 7.3.5 Relevance o f the W H H L Rabbit Model 137 7.4 Proposed Mechanisms for PMio Induced Atherosclerotic Plaque Destabilization... 140 7.5 Proposed Mechanism of PMio Induced Morbidity and Mortality 142 7.6 Concluding Remarks 144 7.7 Future Directions 145 8 Bibliography 146 9 Appendices 165 V I List of Tables Table 1.1. The 2005 daily and annual mean guidelines for particulate matter exposure as recommended by the World Health Organization 7 Table 1.2. Major and minor risk factors for atherosclerosis 16 Table 1.3. Characteristics of stable and unstable atherosclerotic plaques 27 Table 2.1. The composition of EHC-93 urban particulate matter 38 Table 3.1. Major findings from Suwa and colleagues' exposure of W H H L rabbits to PMio .45 Table 3.2. Major findings from Sun and colleagues' exposure of A p o E -/- mice to PM2.5....46 Table 3.3. Sample distribution from the abdominal aorta of W H H L rabbits 48 v i i List of Figures Figure 1.1. Dai ly mortality and air pollution levels in London England during December 1952 2 Figure 1.2. A schematic of particle deposition throughout the respiratory system 11 Figure 1.3. Relative deposition of aerosols in three different regions of the respiratory system in relation to particle size 12 Figure 1.4. Initiation of an atherosclerotic plaque in a muscular artery 20 Figure 1.5. Type II fatty streak atherosclerotic plaque 21 Figure 1.6. Type III atherosclerotic plaque 22 Figure 1.7. Type IV fibrous atherosclerotic plaque 24 Figure 1.8. Advanced type V fibrous atherosclerotic plaque 25 Figure 1.9. Complicated advanced type V I atherosclerotic plaque 26 Figure 2.1. EHC-93 particulate matter 37 Figure 2.2. Tissue processing protocols for transmission and scanning electron microscopy studies 41 Figure 3.1. The three layers of a healthy arterial wall 49 Figure 3.2. Atherosclerotic plaques from control W H H L rabbit abdominal aortas 51 Figure 3.3. Atherosclerotic plaque from PMio exposed W H H L rabbit abdominal aortas 52 Figure 3.4. Macrophage-derived foam cells and S M C derived foam cells in the upper cap region of an atherosclerotic plaque from a PMio exposed W H H L rabbit 53 Figure 3.5. Quantification of the subendothelial population of macrophage-derived foam cells 54 Figure 4.1. Sample transmission electron micrograph from a control plaque with a superimposed sigmoid grid mask 59 Figure 4.2. Dense extracellular matrix subtending endothelial cells 60 Figure 4.3. Fragmented extracellular matrix subtending endothelial cells 61 Figure 4.4. Macrophage in direct contact with the abluminal surface of an endothelial cell .61 Figure 4.5. Macrophage-derived foam cell in direct contact with the abluminal surface of an endothelial cell 62 Figure 4.6. A smooth muscle cell in direct contact with the abluminal surface of an endothelial cell 62 Figure 4.7. Smooth muscle cell derived foam cell in direct contact with the abluminal surface of endothelial cells 63 v i i i Figure 4.8. Unknown cell structures in direct contact with the abluminal surface of endothelial cells 63 Figure 4.9. Space subtending endothelial cells 64 Figure 4.10. The number o f intersects per micrograph analyzed i n the morphometric analysis 65 Figure 4.11. Intima of non-diseased abdominal aorta 66 Figure 4.12. Fibrous cap of a stable plaque in control W H H L rabbit 67 Figure 4.13. Architecture of control atherosclerotic plaques 68 Figure 4.14. Architecture of fibrous atherosclerotic plaques from a PMio exposed rabbit 69 Figure 4.15. Architecture of an atherosclerotic plaque from a PMio exposed rabbit 70 Figure 4.16. Lymphocyte in contact with the endothelium of a control atherosclerotic plaque 71 Figure 4.17. Macrophage-derived foam cells in an atherosclerotic plaque 72 Figure 4.18. A macrophage-derived foam cell at the shoulder o f a control atherosclerotic plaque 73 Figure 4.19. Act in rich smooth muscle cells in control plaques 74 Figure 4.20. Smooth muscle cell derived foam cells 74 Figure 4.21. Smooth muscle cell derived foam cell 75 Figure 4.22. Smooth muscle cell derived foam cell 76 Figure 4.23. Synthetic smooth muscle cells 77 Figure 4.24. Smooth muscle cell orientation in an area o f the media and central core o f an atherosclerotic plaque 79 Figure 4.25. Extracellular l ip id deposition i n the core of an atherosclerotic plaque 80 Figure 4.26. Zebra bodies within macrophage-derived foam cells located in the core of an atherosclerotic plaque 81 Figure 4.27. Relative percentage o f endothelial cell abluminal membrane contacts 82 Figure 4.28. Derivation of a control serial 3-dimensional reconstruction of the abluminal extracellular matrix 84 Figure 4.29. Topographical match between the endothelial basal plasma membrane and the reticulum of electron dense extracellular matrix in control atherosclerotic plaques 85 Figure 4.30. Derivation of a PMio serial 3-dimensional reconstruction of the abluminal extracellular matrix 86 Figure 4.31. PMio 3-dimensional reconstruction of the abluminal extracellular matrix / endothelial contact surface 87 Figure 4.32. Comparison of the control and PMio 3-dimensional reconstructions o f the abluminal dense extracellular matrix 87 Figure 5.1. Atherosclerotic plaque from control W H H L rabbits 101 Figure 5.2. Atherosclerotic plaque from PMio exposed W H H L rabbits 102 Figure 5.3. Absent endothelium in 4% paraformaldehyde fixed atherosclerotic plaque 103 ix Figure 5.4. Positive staining for laminin and type IV collagen in the human prostate 104 Figure 5.5. Immunohistochemistry analysis for laminin and type IV collagen in the W H H L rabbit 105 Figure 6.1. A n illustration of leukocyte rolling, tethering, adhesion and migration I l l Figure 6.2. Surfaces of non plaque endothelium 113 Figure 6.3. Endothelial cell orientation at an arterial bifurcation 113 Figure 6.4. Caveolae on the surface of atherosclerotic plaque endothelium 114 Figure 6.5. Topography of an atherosclerotic plaque 115 Figure 6.6. Cross section of an atherosclerotic plaque from a control W H H L rabbit 116 Figure 6.7. Control atherosclerotic plaque endothelium 117 Figure 6.8. Atherosclerotic plaque endothelium from a PMio exposed rabbit 118 Figure 6.9. A leukocyte cluster over the core of an atherosclerotic plaque from a PMio exposed rabbit 119 Figure 6.10. Endothelial cells protruding into the lumen due to subendothelial macrophage-derived foam cells 120 Figure 6.11. Macrophage-derived foam cell lifting the endothelium up into the blood vessel lumen 121 Figure 6.12. Leukocyte trail 121 Figure 6.13. A n illustration of leukocyte immigration and emigration 123 Figure 6.14. Migrating leukocytes on the surface o f plaques from PMio exposed rabbits... 124 Figure 6.15. Emigrating macrophage-derived foam cell 125 Figure 6.16. Cel l immigration into an atherosclerotic lesion 126 Figure 6.17. Endothelial erosion and subsequent platelet adhesion on exposed extracellular matrix 127 Figure 7.1 Altered plaque organization following PMio exposure 141 Abbreviations A / B / C A H A B A P C R P D/E E C E C M EHC-93 E M T E T F/G /H G M - C S F H & E stain H D L I I C A M - 1 I E L IL J / K / L K4[Fe(CN) 6 ] L D L L P S M/N/O M C P - 1 M - C S F M M P N O American Heart Association Benzo (a) pyrene C-reactive protein Endothelial cell Extracellular matrix Environment Health Canada 1993 (urban air pollution sample) Erin M Tranfield Endothelin Granulocyte macrophage colony stimulating factor Hematoxylin and eosin stain High density lipoproteins Intercellular adhesion molecule-1 Internal elastic lamina Interleukin Potassium ferrocyanide L o w density lipoprotein Lipopolysaccharide Monocyte chemoattractant protein-1 Macrophage colony stimulating factor Matrix metalloproteinase Nitric oxide x i p P A F Platelet activating factor P A H Polyaromatic hydrocarbons P A S Stain Periodic acid-Schiff stain PMio Pollution smaller than 10 um in diameter PM2.5 Pollution smaller than 2.5 um in diameter P M N Polymorphonuclear leukocytes P O M Polycyclic organic matter Q/R R B C Red blood cell R E R Rough endoplasmic reticulum R O S Reactive oxygen species S S E M Scanning electron microscope S M C Smooth muscle cell T T E M Transmission electron microscope T N F - a Tumor necrosis factor-a T B O stain Toluidine blue O stain u/v/w V C A M - 1 Vascular adhesion molecule-1 W H H L rabbit Watanabe heritable hyperlipidemic rabbit W H O World Health Organization X A 7 Z x i i Acknowledgements Thank-you kindly to my advisory committee: Drs. James Hogg, Stephan van Eeden, Bruce McManus and Co l in Fyfe for all their suggestions and support throughout my PhD. A very special thank-you to my graduate supervisor and mentor Dr. David Walker. Without his patience and guidance, I probably would have run away and joined the circus as I threatened to do on many occasions. Dr. Walker gave me guidance when needed, yet the freedom to set my own course through science. There are not words to express my appreciation for everything he has done on my behalf. Thank-you to the members of the James Hogg i C A P T U R E Centre for all their behind the scenes work in maintaining microscopes, laboratory equipment, as well as other equipment and materials I used every day. Thank-you to Claire Smits, the late Sasha Kerjner and the late Diane Minshall for their help with the W H H L rabbit experiments and other animal related questions I had. Thank-you to Dean English, and the late Stuart Greene for imaging expertise as well as Dr. Mark Elliott for his guidance on the immunohistochemistry, particularly the troubleshooting. Thank-you to Renaud Vincent for the E H C - 9 3 particles and his assistance with questions pertaining to the collection and source of these particles. Thank-you to Winnie Enns from the U B C Department of Pathology and Laboratory Medicine, Histology Laboratory for her assistance with sectioning and microanatomical staining. Thank-you to Derrick Home at the U B C Bioimaging Facility for help with S E M imaging of E H C - 9 3 . Thank-you to the ladies in the St Paul's Hospital Library: Barbara Saint, Darlene Bailey, Angela Doyle, and Ani ta Stabrawa for all their help with document delivery and my never ending questions. A n d thank-you to Laura Rossy and Al l en Haddrell for reading sections o f my PhD thesis. x i i i Thank-you to the agencies that supported the research: The Heart and Stroke Foundation of B C and Yukon and The Canadian Institutes o f Health Research. Thank-you as well to Mrs . Cordula Paetzold for fellowship support through the Cordula and Gunter Paetzold Fellowship. I would also like to thank Fanny Chu for her constant help with the T E M and for being such a pleasure to work with, as well as Dr. A l i Behzad for his help with the T E M , the gift o f the E M micrograph and for being a source of constant banter around the laboratory. A special thank-you to my accomplices, also known as fellow graduate students and friends, at the i C A P T U R E Centre, namely Arwen, Rani, Hubert, A l , M c D , Caroline, Wendy, Faye, and Farah for all their support through the tough times and mischief during the fun times. Without them, the process of a PhD would have been much less memorable! Thank-you to my ISU family, James, Tracy, Jo, Adina, JF and A s h for being my cheering squad around the world and breaking the stress by bring laughter induced tears to my eyes. I think we should organize a boat party to celebrate... Thank-you to my dear friends Jenna, Tove and Laura for not filing a missing persons report with the police while I wrote my thesis and for their constant support throughout the 9 years we have been friends. Friends like them make me one of the luckiest people I know. A n d last but certainly not least my parents, Robby and Terry Tranfield, for their support and encouragement. Thank-you to my mom for reading my thesis and finding my odd spelling mistakes (secret versus secrete!). Thank-you to my pesky brother Morgan, my Nana, Grandma T and Mumma for their constant love and encouragement. Thank-you to Aunty Barb and Uncle Harry for their ever present advice, kind words and dinners to feed their graduate student. To those I have not mentioned, your support has always been appreciated. I don't think a PhD is done alone. If it were not for the amazing people in my life, I never would have survived the process and enjoyed it as much as I did. M y sincere appreciation to all! x iv Dedication Some teach, others inspire; I would like to dedicate this thesis to the people in my life who have inspired me and pushed me to be more than I thought I could be. The names are listed in no order and for many different reasons. To each, I simply say thank-you. Stuart Greene Dr. David Walker Robby and Terry Tranfield xv CHAPTER 1 Introduction "If 25 years ago I had said that the majority of Canadians would be drinking bottled water today, you would have laughed. If I were to say now that we may have to breathe bottled air in 25 years if we don't fundamentally change the way our society works, would you laugh? " -Jim Harris 1.1 The History of Air Pollution Every 15 seconds a person dies as a consequence o f air pollution exposure amounting to in excess o f 2 mil l ion annual deaths.1 This conservative estimate by the Wor ld Health Organization (WHO) is based on extensive epidemiological and basic science research that has established a strong positive association between exposure to particulate matter air pollution smaller than 10 pm in diameter and cardiopulmonary morbidity and mortality. 2" 4 This estimate applies only to air pollutants and does not include deaths caused by global warming phenomena such as hurricanes, extensive flooding or droughts. Although there has been increasing focus placed on air pollution related deaths in the past 50 years; the first known environmental legislation originated in Israel. 5 Practitioners o f the Jewish religion suspected a relationship between human illness and the foul odours o f tanneries as well as the airborne waste products from threshing floors. Their laws were documented in text 2000 years ago, though their oral laws are thought to date back an estimated 4000 years.5 These laws stipulated that industries should be located down wind o f cities and towns, minimizing citizen exposure to airborne pollutants. 5 Over 1600 years later, in 1661, John Evelyn wrote the K i n g of England regarding the soot in the air over London. He attributed chronic coughs and pulmonary mortalities to the air quality and he put forward recommendations to move industry outside the city to reduce air pollution. 6 The suggestions of John Evelyn were ignored and air pollution levels continued to 1 Chapter 1: Introduction rise, pushed upwards by the 18th century industrial revolution, the era commonly considered to mark the beginning of our current fossil fuel consuming, energy dependent economy. Yet, it wasn't until the 20 t h century that a series of events focused the attention o f the medical and scientific communities on air pollution related morbidity and mortality. In 1930, 63 people died during an air pollution episode in the Belgian Meuse Val ley . 7 In 1948, 20 people died, several animals died and 43% of the 14,000 inhabitants became i l l after a weather inversion in the highly industrialized town of Donora, Pennsylvania trapped coal smoke, sulphur dioxide, soluble sulphates, and fluorides over the city. 8 Yet, the event cited as the major turning point in air pollution awareness and public policy did not occur until 1952. In December of that year, London, England suffered an absence of wind and a temperature inversion resulting in a very dense fog over the Greater London area. Particulate matter produced by the burning of coal was trapped within the fog, leading to a rapid and extreme rise in air pollution followed closely by a rapid and extreme rise in mortality rates (Figure 1.1). During the four day pollution event in excess of 4000 deaths above typical levels occured. 9 In response to the startling number of air pollution related deaths, the British Parliament passed a Clean A i r Ac t in 1956 to reduce emissions of airborne pollutants. 1 0 Their actions were validated when in 1962 the meteorological conditions of London were similar to those experienced in December 1952, yet the increase in particulates was not as extreme, and even with the increased population of London, the rise in mortality was only a sixth of that experienced in 1952 . 1 0 , 1 2 Seven hundred additional deaths in 1962 were still o f concern, yet a drastic improvement over the 4000 experienced ten years earlier. 600 >500 & 1300 200 100 Deaths Pollution 3000 2400^ 1 1800 I EL B 1200 42 J 600 I 0 15 5 10 Day Figure 1.1. Daily mortality and air pollution levels in London England during December 1952. Adapted from Schwartz 1994. 1 1 2 Chapter 1: Introduction Research on air pollution related morbidity and mortality began in the 1950's and has steadily increased. The 50,000 scientific articles on air pollution provide undeniable epidemiological evidence that air pollution exposure has a strong association with cardiopulmonary fatal events. What remain largely unknown are the cellular mechanisms responsible for the physiological effects associated with air pollution exposure. The focus o f this thesis dissertation was to investigate changes induced at the tissue and cellular levels as a consequence of air pollution exposure. 1.2 The Causative Agent: Particulate Matter Air Pollution A i r pollution is a complicated mixture of airborne particulates combined with mist and fog, and gases such as ozone, and carbon monoxide . 1 3 ' 1 4 Since 1952, the search for the causative agent of air pollution related mortality has been pursued and gradually narrowed down to particulate matter smaller than 10 um in diameter (PMio) . 1 5 In 1979, an extensive report was written by a group o f prominent British scientists suggesting airborne particulates were responsible for some of the observed cardiopulmonary health effects. 1 0 Nonetheless it was not until a standardized definition of particulate matter was introduced in 1987 by the United States Environmental Protection Agency, 1 6 and statistical analysis was used to remove major confounding factors including smoking, socioeconomic status, and body-mass index that the first robust positive association between mortality and particulate matter air pollution was established in 1993. Gaseous atmospheric compounds, which at high levels are considered pollutants, such as ozone, carbon monoxide, sulphur dioxide and nitrogen dioxide have been shown to have some positive association with morbidity and mortality. 1 7" 2 0 However, those observed associations are much less robust than the association consistently observed for particulate matter induced morbidity and mortal i ty. 4 ' 2 0 2 3 Particulate matter is defined as a stable atmospheric suspension of solid and liquid particles with variable composition, origin and an aerodynamic diameter ranging from -0.002 to -100 u m . 1 4 ' 2 4 The lower size limit to particulate matter remains undefined as the chemical process and size spectrum at which a mass o f molecules condenses into a particle is as yet undefined. 1 4 The upper size limit to particulate matter corresponds to the particle size that settles rapidly out of the lower atmosphere because of the influence o f gravity. 1 4 Particles 3 Chapter 1: Introduction with an aerodynamic diameter smaller than 10 um are the subset of monitored particles because of their connection to human morbidity and mortality. 2 5 Prior to the establishment of the PMio classification by the Environmental Protection Agency in 1987, many different 26 terms were used to describe biological relevant airborne particles, including British smoke, 77 78 7Q total suspended particles, ' and coefficient of haze. ' A 1994 paper by Dockery and Pope 2 5 established conversion factors between P M i o and previously used terminology thus completing a standardization process of terminology in the field o f particulate matter air pollution. Particulates are formed by two main processes. The first process is the condensation of gases in the troposphere. 1 3 ' 1 4 The second process is mechanical such as wind blowing over crustal surfaces or human activities in construction sites, agricultural industries and resource harvesting industries.3' 1 3 , 1 4 ' 3 1 O f all potential sources, human activities, namely the combustion of fossil fuels in industry, transportation and energy production are by far the greatest generator of PMio through both the condensation and mechanical processes . 1 3 ' 1 4 ' 3 1 Source variability and formation processes of particulate matter affect not only size distribution but also particle composition. 3 2" 3 4 1.2.1 Particle Size Particle size is thought to be the primary variable in determining biological consequences of pollution exposure. 1 4 ' 3 5 Particulates are generally assumed to be spherical in shape. Particle size is described using the term aerodynamic diameter, which is defined as the diameter of a spherical particle with a density of 1 g/cm 3 (the density of water) that has the same settling velocity as the particle under consideration. 1 3 ' 1 4 Size has been shown to determine three essential variables: 1. the site of particle deposition in the lungs , 3 6 ' 3 7 2. the surface area-to-volume/mass ratios, 3 7" 3 9 (see Appendix I for terminology definition) and 3. the rate at which the particles are cleared from the lungs. 3 7 Particles larger than 10 um in diameter are mainly generated by wind activities over soi l . 3 These particles do not pose a great danger to human health because they penetrate no deeper than the nasopharynx. 4 0 The inertia of large particles prevents them from turning the tight 4 Chapter 1: Introduction corners of the airway. 3 6 A s a result these particles run into the mucous covered walls of the internal nares and nasopharynx where they become trapped. This process is called inertial impaction. Particles trapped in the upper airway are expelled by clearance through the nose or mucociliary clearance to the back o f the nasopharynx followed by swallowing into the 9S gastrointestinal system. Particulates smaller than 10 um in aerodynamic diameter have a small enough inertia that they can navigate the upper airway and be inhaled through the trachea, bronchial tree and into the alveoli. Particle deposition on the alveolar surface is linked to cardiovascular morbidity and mortal i ty. 3 ' 4 1 A s a consequence, the particulate matter regulated by the United States Environmental Protection Agency is PMio . Analysis o f PMio has revealed three distinct fractions: the coarse fraction, the fine fraction and the ultrafine fraction. Each fraction has distinctly different formation processes, composition, atmospheric lifetimes and biological consequences.3 8 The coarse fraction contains particles between 10 and 2.5 um in aerodynamic diameter that are produced by mechanical processes such as wind erosion. These larger particulates are the fraction most affected by gravitational settling and typically settle out of the lower atmosphere within hours of formation. ' The coarse fraction is thought to penetrate as deep as the pharynx and occasionally into the trachea. The primary sites of deposition for coarse particles are the posterior surface of the nasopharynx, the larynx and the carina o f the t rachea . 3 5 ' 4 2 ' 4 3 The coarse fraction is carried up the trachea by the mucociliary escalator and swallowed into the gastrointestinal system. Similar to the >10 pm fraction, particles deposited in the nasopharynx are expelled by coughing or by mucociliary clearance and swallowing. The fine particles, also known as the accumulation mode, are between 2.5 and 0.1 um in size. These particles account for the greatest mass of airborne particulates and are generally formed through human activities. 1 3 Fine particle formation typically occurs when gas molecules condense together to form particles through heterogeneous and homogeneous nucleation, as well as condensation onto already existing atmospheric p a r t i c l e s . 1 3 , 1 4 ' 3 8 The 5 Chapter 1: Introduction small size of this fraction of particles makes them less susceptible to gravitational settling resulting in atmospheric lifetimes in the order of days, 1 3 and the ability to be transported long distances by wind currents. The fine particles are inhaled into the conducting airways o f the lungs. A s the combined airway cross section increases, the flow velocity in the conducting airways decreases resulting in particle sedimentation. Particles deposited in these areas are cleared by the mucociliary escalator. Particles that are not deposited in the conducting airways reach the alveoli where they diffuse outwards, eventually contacting the surfactant layer of the alveolar wall . In the alveoli, a population o f resident macrophages patrol the alveolar surfaces immediately below the layer of surfactant. Particles deposited on the alveolar wall are phagocytosed by these alveolar macrophages. The macrophages are then typically transported up the conducting airways and trachea by mucociliary clearance or taken to the pulmonary lymph nodes. 4 4 A small population of free particles that were not phagocytosed by alveolar macrophages may translocate into the lung interstium, and be taken up with interstitial fluid into the lymphatic vessels for processing in the pulmonary lymph nodes. 2 5 Depending on their solubility, particles may have a half life ranging from several days to thousands of days within the pulmonary lymph nodes 4 5 The smallest fraction of particulate matter is composed of ultrafine particles which are smaller than 0.1 pm in aerodynamic diameter. These particles have traditionally been considered the fresh emissions in pollution that have yet to undergo condensation or modification processes. The composition of fine particles is varied, characteristically composed of ammonium, carbon, nitrate and sulphate as well as trace metals formed in the 10 combustion processes. This fraction of the smallest particles accounts for the greatest number of atmospheric particles with the largest surface area to mass ratio. 2 4 ' 3 8 These particles are primarily deposited on the alveolar surface by diffusion through the a i r . 3 6 ' 4 2 ' 4 3 Smaller particles are also believed to translocate into the pulmonary circulation 4 6 as evidenced by their eventual accumulation in the lymph nodes, 4 4 ' 4 5 spleen, 4 7 heart, 4 7 l i v e r 4 8 ' 4 9 and even the bladder 4 6 and brain. 5 0 6 Chapter 1: Introduction The large surface area to volume/mass ratios o f fine and ultrafine particles may account for their increased deleterious impact on human health in a number o f ways. Evidence in the 37 51 literature suggests that the larger the surface area the greater the impact o f the particle. ' 37 Smaller particles are known to have the greatest surface area relative to their mass as well 38 as the greatest efficiency at penetrating deeply into the lungs. In response to epidemiological investigations revealing that the fine and ultrafine fractions have the strongest associations with morbidity and mortality, 3 3 ' 5 2 5 4 the Wor ld Health Organization recently updated the daily and annual mean guidelines of acceptable air pollution (Table l . l ) . 5 5 The detrimental health effects of the PM2.5 fraction resulted in stringent guidelines. 5 5 Within a 24-hour period, the mean level of PM2.5 should not exceed 25 pg/m 3 and the mean level of PMio should not exceed 50 pg/m 3 . Over the course of a year, the mean level of PM2.5 should not exceed 10 pg/m 3 and the mean level of PMio should not exceed 20 pg/m . Mean standards per 24-hours are higher than mean standards per year to allow for infrequent air pollution events caused by non controllable factors such as wi ld fires, or weather pattern changes. Data gathered prior to 2000 suggest that most major American cities would not have met these updated standards;5 6 however, PMio levels are gradually decreasing with the implementation of pollution minimization measures. Table 1.1. The 2005 daily and annual mean guidelines for particulate matter exposure PM2.5 10 iig/m3 annual mean 25 pg/m3 24-hour mean PMio 20 pg/m3 annual mean 50 ug/m3 24-hour mean 1.2.2 Particle Composition The composition of PMio in the troposphere is dependent on the environment in which the particles were made, the process which made the particles and any post formation surface reactions that may have occurred. 1 4 This results in large composition variability of particulates between cities, and even between districts within a ci ty. 2 4 ' 5 7 ' 5 8 Particle 7 Chapter 1: Introduction composition is currently not considered a significant determinant of morbidity and mortality because when analyzed on a global scale, particles of varying composition, yet similar sizes have strikingly similar physiological effects. 5 9 This repeated observation has led to the assumption that size rather than composition is the causative variable in air pollution related morbidity and mortality. There is the concern that epidemiological sampling methods may be leading to a biased perspective. The inherent challenge to epidemiological studies is the reliance on relatively few pollution sampling stations as an indicator of particle size, concentration and composition across entire cities. 1 7 Furthermore, individuals can have different daily habits, such as long commutes in traffic, long periods of time indoors or outdoors, or a residence near pollution sources. These behavioural differences makes personal exposure highly variable between individuals. If changes in composition have subtle effects on cardiovascular morbidity and mortality, the positive correlation may not be detected using minimal sampling stations over a large area. These sampling limitations may have led to an incorrect consensus that size is of greater significance. A s such, the biological consequences of particle composition merit exploration until particle composition has been conclusively shown to not be associated with cardiovascular related morbidity and mortality. Particle composition is characteristically a combination of both organic and inorganic materials with organic making up 10 - 40% of the total particle mass. 3 4 Carbon is typically the largest component of particles with various combinations o f inorganic heavy metals and toxic elements making up the remaining mass. 6 0 Elements such as Ca, Ba , Fe and M n are derived from the earth's crust and have relatively low toxicity. 3 4 Other elements such as A s , C d , Cr, Cu , Hg , N i , Pb, V and Zn, which with the exception of A s are all transition metals (see Appendix I for terminology definition), are of greater concern. Several studies have shown that PM2.5 has a higher toxicity and this is thought to be due in part to the transition metals on the surface of the particles. 6 1 Findings such as these have led to the Wor ld Health Organization placing exposure limits on A s , Cd , M n , N i , and V . 6 2 In addition, to this, Pb content has been regulated by the United States Environmental Protection Agency since 63 1994. Each element can typically be tied to specific anthropogenic activities. For example, 8 Chapter 1: Introduction Z n has been shown to be a by-product of vehicle tire deterioration on highways; ' C u is associated with industrial areas, particularly diesel engines; 6 6 ' 6 7 and N i is mainly associated with fossil fuel use, oi l burning, and emissions from stationary and industrial sources . 3 4 ' 6 7 ' 6 8 Different cities with different industries w i l l have varying levels o f contaminants in their associated troposphere. A s a direct result of this variability, particles w i l l vary in composition between regions and even within regions. ' The organic component of PMio has also been shown to have an enhanced effect, particularly polyaromatic hydrocarbons ( P A H ) 6 9 and endotoxins. 6 1 P A H are compounds produced by the incomplete combustion of organic materials such as wood, vegetation, meat, coal, fossil fuel, garbage, and tobacco. 6 9 ' 7 0 Following incomplete combustion, complicated mixtures of P A H are released as polycyclic organic matter ( P O M ) . 1 4 Individual P A H are composed of ring structures. The smaller ring compounds, such as the two ringed compounds, remain gaseous at normal temperatures; the larger four or five ringed P A H condense into solids, attaching themselves to atmospheric particles. 1 4 ' 7 0 Combustion processes tend to generate smaller particles along with the P O M mixtures, thus the P A H percentage in the fine and ultrafine particle fraction is much higher than it is in the coarse fraction. 6 9 The United States Environmental Protection Agency has identified 16 cancer causing P A H compounds, 70 particularly benzo (a) pyrene (BaP) which is the most toxic P A H . The danger to human health stems from the high concentration of cancer causing P A H compounds in the fine and the ultrafine fractions. P A H have been extensively studied in the industrial setting resulting in established exposure l i m i t s . 6 9 ' 7 1 However, non-industry P A H exposure has been the focus of limited research and exposure limits have yet to be established. The effect of the P A H component of PMio on cardiopulmonary morbidity and mortality is an area of ongoing research. Endotoxins are a product of gram-negative bacteria and are potent stimulators of pro-inflammatory gene expression, reactive oxygen species (ROS) and lipid mediator release. Endotoxins in PMio samples have been shown to stimulate endotoxin receptors on the surface o f alveolar macrophages. 7 2 ' 7 3 L P S coating increases the translocation of ultrafine particles when compared to uncoated ultrafine particles. 7 4 In the presence of L P S there is a 9 Chapter 1: Introduction stimulation of an excessive response to PMio exposure and an increase in tumor necrosis factor (TNF)-a release. 7 5 The study of Imrich and colleagues 7 5 suggests that complex interactions between overly sensitized macrophages and lung epithelial cells resulted in a heightened local response and potentially a heightened systemic response. A s with the P A H component of PMio , the endotoxin component is an area of ongoing research. Both organic constituents have strong circumstantial associations with a heightened response to PMio ; ongoing research is seeking direct evidence. 1.2.3 Particle Shape In addition, to size and composition, the shape of particles warrants mentioning. There is little research regarding shape, perhaps because the general assumption has been made that particles are spherical. ' Contrary to this assumption, imaging of particulates reveals particles with irregular shapes, in most cases appearing to be agglomerates o f many smaller part icles . 1 3 ' 1 4 A n irregular shape would have a larger surface area than a spherical shape. A greater particle surface area is known to be more toxic to the lungs . 3 7 ' 5 1 In addition, it may have altered dispersion or settling patterns to spherical particles. The literature on the consequence of shape is scarce, leaving many unanswered questions. However, with the recent design of techniques to accurately, reliably and rapidly determination particle shape, the field of particle shape w i l l be explored in upcoming years. 1.2.4 Particle Dispersion in the Lungs The dispersion of particulate matter throughout the lungs and the location of deposition of the particulate is important in observed physiological changes. 7 7 Yet, there are many variables that affect the extent and location of particle deposition within the lungs such as particle size, density and shape; airway geometry; and breathing pattern o f the exposed i n d i v i d u a l . 1 3 ' 4 2 ' 4 3 Smaller particles, specifically ultrafine particles are more readily deposited in the deep regions of the l u n g s . 4 2 ' 4 3 ' 7 8 Posture in combination with gravity are important determinants of primarily sedimentation deposition in the lungs. 3 6 In addition, depth of breathing is an important variable for the mixing, dispersion and deposition of particles throughout the lungs. 7 9 Physical exercise results in increased particle deposition in the lungs, presumably 10 Chapter 1: Introduction from increased minute ventilation. 8 0 Furthermore, exercise results in a greater pulmonary diffusion capacity enabling the rapid exchange of gases,8 1 and perhaps increased translocation of ultrafine particles. Further research is needed to address questions regarding variable susceptibility of specific areas of the lungs, and the possibility of an altered physiological effect of particles i f deposition occurs in several large deposits versus many small scattered deposits. Coarse particle deposition ( 1 0 - 2 . 5 fjm) in the internal nose and trachea Fine particle deposition (2.5 - 0.1 pm) throughout entire respiratory tract, including the alveolar surface Ultrafine particle deposition (< 0.1 pm) throughout entire respiratory tract, including the alveolar J surface, possibility of translocation Figure 1.2. A schematic of particle deposition throughout the respiratory system. Coarse particles are deposited in the upper airway and may penetrate as deep as the trachea. Fine particles penetrate down to the alveolar surface. Ultrafine particles are deposited on the alveolar surface and there is some speculation o f translocation into the systemic circulation (diagram by Er in Tranfield, pulmonary system diagram modified from Van De Graaff 2002 8 2 ). 1.2.5 Existence of a Threshold Epidemiological investigations suggest that there is no threshold for adverse cardiopulmonary events resulting from air pollution exposure but rather that air pollution is deleterious to human cardiopulmonary health at all levels. 8 3" 8 7 Studies from different laboratories, on different continents all clearly show a linear association between the amount of inhaled particles and the risk of acute cardiopulmonary events . 1 1 ' 2 6 ' 8 7 11 Chapter 1: Introduction Though cardiopulmonary mortalities have a linear association to PMio exposure, there appears to be a threshold of 50 pg/m 3 for all other causes of mortality. PMio exposures above 3 • 83 the 50 pg/m concentration has a subsequent linear relationship with all causes o f mortality. Relative Deposition Nasal Alveolar Tracheo-bronchial 100 30 10 3 1 0.3 O'.l 0.03 0.01 A e r o s o l Diameter (pim) Figure 1.3. Relative deposition of aerosols in three different regions of the respiratory system in relation to particle size. Diagram modified from Hlastala 2001. 4 0 1.2.6 Lag Period The existence and extent o f a lag period between exposure and observed cardiopulmonary acute events is still an area of contention requiring further research. 8 4 ' 8 5 However, the current literature suggests that response curves are very similar whether a zero day, one day, or two day delay is factored i n 8 3 , 8 6 suggesting there is no lag period. 1.3 Morbidity and Mortality associated with PMi0 Exposure Epidemiological studies present robust links between ambient PMio and cardiopulmonary mortality. 2" 4 Because of global widespread cardiovascular disease burden, 1 cardiovascular 12 Chapter 1: Introduction events following PMio exposure account for the greatest proportion of pollution related morbidity and m o r t a l i t y . 1 1 ' 8 8 - 9 0 Specifically the incidences of myocardial infarct ions, 9 0 ' 9 1 strokes, 1 1 ' 9 2 heart failure exacerbations, 9 0 cardiac arrhythmias 9 3 and sudden deaths 1 1 increase within hours o f exposure to elevated levels o f PMio . Although epidemiological studies can not prove causation, they can establish positive associations and suggest targets for mechanistic studies to investigate specific pathophysiological responses that may be occurring as a consequence of ambient particulate matter exposure. From a pathophysiological standpoint, the link between PMio exposure and cardiovascular related morbidity and mortality remains perplexing. Human studies have provided evidence that PMio exposure results in a decrease in heart rate variability, 9 4 and an increase in circulating white cells , 9 5 heart rate, 9 6 cardiac arrhythmias, 9 3 systolic blood pressure, 9 7 plasma viscosity, 2 8 arterial vasoconstriction, 9 8 and the procoagulant and proinflammatory state o f the b lood. 9 9 In animal studies, exposure to PMio affects heart rate and blood pressure, 1 0 0 and increases arrhythmias, 1 0 1 fibrinogen levels , 1 0 2 platelet activation, 1 0 3 and results in early release of polymorphonuclear neutrophils 1 0 4 and monocytes 1 0 5 into the circulation. Taken together, these findings suggest that exposure to particulate air pollution initiates a systemic inflammatory response that may predispose the exposed individual to many of the physiological parameters associated with fatal cardiovascular events. A previous report by Suwa and colleagues 1 0 6 from our laboratory provided light microscopic data showing morphological changes in atherosclerotic plaque structure consistent with less stable atherosclerotic plaques from Watanabe heritable hyperlipidemic ( W H H L ) rabbits exposed to PMio. The study provided evidence o f increasing plaque size, extracellular and total l ipid deposition, E C M deposition and monocyte recruitment into atherosclerotic plaques as a consequence of PMio exposure. Using immunohistochemistry and functional studies Sun 107 and colleagues reported increased lipid deposits and macrophage invasion into the intima and media o f atherosclerotic plaques, altered vasomotor tone, and increased atherosclerosis burden in an A p o E -/- mouse model following a long-term exposure to low concentrations of PM2.5- These studies used light microscopy to suggest changes in atherosclerotic plaque size and composition as a consequence of P M i o exposure. What remain unknown are the possible 13 Chapter 1: Introduction mechanisms by which particulate air pollution exposure mediates the physiological changes outlined in the previous paragraph and how these changes result in acute cardiovascular events and sudden death. Epidemiological investigations have identified several at-risk populations. Particulate air pollution exposure has been repeatedly linked to adverse events in individuals with a prior myocardial infarction 8 8 ' 1 0 8 - 1 1 0 or chronic diabetes, 1 1 1 both o f which are associated with advanced atherosclerotic disease. Furthermore, exposure to PM2.5 had a significant effect in A p o E -/- mice fed high fat chow, where as a diminished effect was observed in A p o E -/-mice on regular chow diets. 1 0 7 Collectively these observations suggest that particulate matter exposure has a deleterious impact in combination with high l ipid diets, and a heavy atherosclerotic burden. Atherosclerotic plaque disruption with thrombus formation is the predominant mechanism leading to unstable angina, myocardial infarctions and sudden dea th . 1 1 2 ' 1 1 3 Therefore, it seems likely that air pollution related morbidity and mortality may also be associated with advance atherosclerotic disease and plaque disruption. 1.4 Atherosclerosis Atherosclerosis is a disease of the medium and large blood vessels, typically characterized by fatty deposits under the endothelium of the vessel wall and commonly considered to be an inflammatory disease. 1 1 4 ' 1 1 5 Atherosclerosis is not a new disease; studies on mummies from the Egyptian era have proven that atherosclerosis is a disease that has affected human kind for thousands of years, 1 1 6 ' 1 1 7 although it is only in recent times that this disease has accounted for such a substantial percentage of the global burden of disease. 1.4.1 Global Burden of Disease In excess of 30% o f all global deaths are attributed to cardiovascular disease making it the leading cause of death worldwide and accounting for more than 17 mil l ion deaths every year.1 Though a human life can never be measured in economic terms, the treatment of a single person with coronary artery disease was conservatively predicted to cost $82,000 U S 14 Chapter 1: Introduction in 1996.1 When inflation is factored in and applied to the number of patients with coronary artery disease, the economic impact of atherosclerosis is enormous. Frequently it is assumed that cardiovascular disease affects affluent countries whose citizens can indulge in lavish lifestyles. Contrary to those expectations, W H O estimates show that the three countries with the greatest cardiovascular mortality rates are Russia, China and India. 1 Countries such as Australia, the United States o f America, Denmark and the Netherlands have had reductions in cardiovascular death rates mostly due to improved prevention, diagnosis and treatment of cardiovascular diseases. Mortality rates between countries vary primarily on prevalence of cardiovascular risk factors and W H O estimates show that obesity, smoking and sedentary lifestyles are now dramatically on the rise in low and middle income countries. It is estimated that cardiovascular disease prevalence w i l l continue to increase globally and 82% of that increase wi l l occur in developing countries.1 There are many biological mechanisms inhibiting the development of atherosclerosis. Under normal healthy conditions the endothelium is impermeable to a vast array o f atherogenic factors. Furthermore, the liver produces high density lipoproteins ( H D L ) to remove low density lipoproteins ( L D L ) from the tissues and circulating blood while the endothelium produces vasoregulatory cytokines to control blood pressure, blood velocity and there-by shear stress (see section 1.4.5 for detailed description of shear stress), acting on the vessel walls. Within the blood vessel wall , tissue monocytes, also called macrophages, clean up any unwanted debris, cells, and lipids, while smooth muscles cells produce collagen, proteoglycans and elastin to provide stability and strength. Under the right circumstances these biological mechanisms can stop atherosclerotic plaque development and even diminish atherosclerotic plaque size. However, 80 - 90% of people dying from cardiovascular disease have at least one o f the major risk factors1 that w i l l counteract the protective biological processes of the body and result in the slow, silent progression o f atherosclerosis. Combine this with a possible genetic predisposition to heart disease, high levels of iron in men and post-menopausal women when compared to menstruating women, and now air pollution and it is understandable why cardiovascular diseases are the leading cause of death in the world. A s the leading killer globally, considerable research is being done to understand the 15 Chapter 1: Introduction pathogenesis of atherosclerosis to facilitate further improvements in prevention, early diagnosis and effective treatment. 1.4.2 Risk Factors for Atherosclerosis Though there are in excess of 300 factors with a positive association to cardiovascular diseases, a select few met the three criteria required by the W H O to qualify as a major risk factor. The three criteria are 1. a high prevalence in many diverse populations; 2. a significant independent impact on the risk of coronary heart disease or cerebral infarction; and 3. treatment and control result in reduced risk. 1 Based on those criteria, the major modifiable risk factors for cardiovascular diseases are smoking, a sedentary lifestyle, high blood pressure and high blood l ipid levels. 1 There are also constitutional, or non modifiable risk factors, which include age, sex and genetics (Table 1.2). 1 1 8 Stress, mental illness, infections, alcohol intake, and low socioeconomic status have also been linked to cardiovascular 1 118 diseases though not to the same extent as the major risk factors. ' Table 1^^ -Smoking - A sedentary lifestyle -High blood pressure -High blood l ipid levels -Male sex -Advancing age -Genetics -Stress -Mental illness -Low socioeconomic status -Alcohol intact -Chlamydia pneumoniae -Stress -Trans fat intake Multiple risk factors have a synergistic effect; two major risk factors increase the risk of an atherosclerotic event by four times, three major risk factors increase the risk by seven t imes. 1 1 8 However, an absence of major risk factors does not preclude an individual from developing atherosclerosis. 1 1 9 1.4.3 A Brief History of Our Understanding of Atherosclerosis Though atherosclerosis has affected humankind for centur ies 1 1 6 ' 1 1 7 it wasn't until the 1800s that scientists began to investigate the pathophysiology o f the disease. The presence of 16 Chapter 1: Introduction inflammation around atherosclerotic plaques was recognized early and became the foundation for a heated controversy between the German pathologist Rudolf Virchow and the 1 9fi Austrian pathologist Carl von Rokitansky. Rudolf Virchow described the atherosclerotic fatty streak and suggested that inflammation was a fundamental player in the initiation and progression of the disease. In contrast Carl von Rokitansky acknowledged the presence of inflammation; however, he viewed inflammation as secondary to atherosclerotic plaque development. The Canadian, Sir Wi l l i am Osier published a medical textbook in 1892 titled 1 9 1 "The Principles and Practice of Medicine" in which he described "arterio-sclerosis: a condition of thickening" in the intimal wall , the onset o f which is dependent on the "quality of the arterial tissue the individual had inherited and the amount o f wear and tear he has subjected it to". Osier suggests other risk factors for atherosclerosis, including stress, syphilis infection, chronic intoxication, over-eating, and overworked vascular muscles due to high 191 blood pressure. In 1908, the Russian scientist Alexander Ignatowski showed that when 199 rabbits were fed eggs and milk atherosclerotic plaques formed thus initiating the long standing theory of l ipid involvement in atherosclerosis. James Herrick published a landmark article in 1912, describing among a long list o f important observations, calcium deposits in partially occluded coronary arteries as well as clots fully occluding blood flow.123 In 1949, JB Duguid showed the incorporation of fibrin into the vascular wall and suggested the involvement of thrombosis in atherosclerosis. 1 2 4 In 1958, Russell Holman, put forward the idea of a stepwise progression of atherosclerotic plaques. 1 2 5 In the 1970s, Russell Ross proposed the Response to Injury Model which centered around the idea that atherosclerosis forms in response to continued injury to and loss of the endothelium. 1 2 6 Since the 1970s, the Response to Injury Model has evolved shifting the focus from an absent endothelium to a damaged endothelium. In the damaged state, the endothelium was thought to allow l ipid and cells to accumulate in the intima becoming the l ipid and cellular deposits characteristic o f atherosclerosis. 1 1 5 The involvement of thrombosis in the initiation of atherosclerosis was also suggested, along with a role for viral infections. 1 2 7 Extensive research and growing understanding of atherogenesis has continued to support the Response to Injury Model , incorporating aspects of the l ipid model and other developing ideas into one central model: atherosclerosis is a chronic inflammatory disease that takes decades to become clinically re levant . 1 1 4 ' 1 2 8 " 1 3 0 17 Chapter 1: Introduction 1.4.4 Pathogenesis of Atherosclerosis: A Chronic Inflammatory Disease This process of plaque development and evolution has been broken down into a series o f steps that begins with elevated levels of circulating L D L , and an abundance of free radicals in the blood caused by smoking, hypertension, diabetes mellitus, and possibly even infectious microorganisms. 1 3 1 In response to the L D L and free radicals, the endothelium maladapts by upregulating the expression of adhesion molecules such as intercellular adhesion molecule ( I C A M ) - l , 1 3 2 vascular adhesion molecule ( V C A M ) - l , 1 3 2 ' 1 3 3 as well as E and P-se lec t in 1 3 2 ' 1 3 4 making it possible for leukocytes and platelets to adhere to the leaky endothelium and monocytes to readily migrate into the blood vessel intima. ' ' Once within the blood vessel wall , the recruited leukocytes, monocytes and T-cells, may become further stimulated by the environment, particularly by the oxidized lipids that have accumulated in the intima, and the monocytes w i l l transform into activated macrophages. The activated cells release chemokines, many of which play central roles in new atherosclerotic plaque development. Interleukin ( IL)- l and T N F - a stimulate further leukocyte adhesion to the endothelial 118 surface. Monocyte chemoattractant protein-1 (MCP-1) is a strong stimulus for leukocyte recruitment, particularly monocyte recruitment, into the i n t i m a 1 1 4 ' 1 3 7 and IL-8 is a powerful neutrophil chemoattractant.1 3 7' 1 3 8 If the initial insult continues, endothelial dysfunction results. Historically, the term endothelial dysfunction has referred to numerous pathological conditions, including impaired regulation o f vascular growth, dysregulation of vascular remodelling and altered anticoagulant and anti-inflammatory properties of the endothelium. Currently, the most common use of the term endothelial dysfunction is in reference to decreased bioavailability of nitric oxide by the endothelium leading to an impairment of vasorelaxation of the vessel w a l l . 1 4 0 To some degree, all o f these pathological changes occur in the endothelium at the onset of atherosclerotic plaque development. Under a constant stimulus such as hyperlipidemia, hypertension, and the physiological consequences of smoking, plaque formation begins to propagate itself; as cellular and l ipid infiltration occurs it leads to endothelial dysfunction and increased endothelial permeability which allows continued cellular and lipid infiltration. Atherogenesis is a progressive, destructive cycle of injury and response leading to further injury. The end result is a complicated spectrum of atherosclerotic plaques that develop over the course of decades. 18 Chapter 1: Introduction 1.4.5 Shear Stress and Atherosclerotic Plaque Development and Progression The risk factors for atherosclerosis, including smoking, a sedentary lifestyle, high blood pressure and high blood lipid levels, act systemically; however, atherosclerosis typically develops in predictable, focal areas . 1 1 5 ' 1 4 1 B y nature of the viscosity of blood, blood flow produces frictional forces on the vessel wall known collectively as hemodynamic shear stress. 1 4 2 Recent studies have confirmed the link between hemodynamic forces and blood vessel structure, endothelial cell function and gene expression. 1 4 2 ' 1 4 3 Hemodynamic stress results in the expression of atheroprotective genes and the maintenance of a healthy vessel w a l l . 1 4 2 In atherosclerotic prone areas, such as the upstream side of vessel bifurcations, the hemodynamic shear stress is lower resulting in turbulent blood flow. The absence of typical flow patterns and shear stress results in altered gene expression leading to an atherogenic endothelial phenotype. 1 4 2 A s atherosclerotic plaques develop, the flow o f blood is increasingly disrupted leading to greater turbulence and further altered gene expression. A n absence of or alteration in shear stress is thus very important and predictable in dictating the site of atherosclerotic plaque format ion . 1 4 2 ' 1 4 4 1.4.6 Pathological Classification of Atherosclerosis The blood vessel wall is composed of the luminal intima, the muscular media and the outer supporting, connective tissue rich adventitia. 1 4 5 The intima is primarily affected by atherosclerosis. The organization of a healthy unaffected arterial intima consists of a layer o f endothelial cells separating the circulating blood from the arterial wal l . The endothelial cells are supported on their abluminal surface by a discontinuous thin basal lamina, and separated from the media by a longitudinal layer of dense elastin called the internal elastic lamina. 1 4 5 Sparse l ipid accumulation and sporadic monocytes and smooth muscles cells are common in the normal healthy blood vessel wall ; however, the threshold at which l ipid and monocyte accumulation within the intima becomes atherogenic is u n k n o w n . 1 4 5 ' 1 4 6 In order to standardize the description of cardiovascular plaques, the American Heart Association ( A H A ) has devised a pathological classification system of six levels of increasing sever i ty . 1 2 7 ' 1 4 6 The first three classifications of atherosclerotic plaques are termed early atherosclerotic plaques and entail negligible disruption o f either the media or the intima. 19 Chapter 1: Introduction These classifications represent a progression from very small and sporadic lipid deposition and mononuclear cell recruitment to extensive and layered lipid deposition and mononuclear cell recruitment. Abnormal, sparse intimal deposits of lipoproteins, particularly LDL and the invasion of the intima by t-cells and by monocytes that become macrophages within the intima is classified as a type I plaque (Figure 1.4).147 This is the earliest form of recognizable atherosclerosis.127 Type I plaques can only be identified using microscopy and/or chemical analysis, and as a consequence they have only recently been studied. Figure 1.4. Initiation of an atherosclerotic plaque in a muscular artery. Under the conditions of inflammation, hyperlipidemia, and hypertension, lipid accumulates in the subendothelial intima and monocytes adhere to and transmigrate through the endothelium. Under continued injurious stimuli, lipoproteins and monocytes / macrophages continue to accumulate in the intima. Sequestered L D L is highly susceptible to oxidation and macrophages express scavenger receptors on their surfaces that have a high affinity for oxidized LDL. This results in the lipid being taken up by the macrophages, activation of the macrophages and eventual transformation of the macrophages into newly formed intimal macrophage-derived foam cells.1 4 8 The list of atherogenic cytokines activated macrophages 20 Chapter 1: Introduction release is extensive and includes in ter leukin ( I L ) - l a , I L - 6 , I L - 1 0 , granulocyte macrophage co lony-s t imula t ing factor ( G M - C S F ) , macrophage colony-s t imula t ing factor ( M - C S F ) , T N F -a, and T N F - p . 1 3 7 In response to continued L D L deposi t ion and macrophage invas ion into the in t ima, macrophage-derived foam cel ls organize into adjacent l a y e r s . 1 2 7 L i p i d accumulates i n the few in t imal smooth muscle cel ls ( S M C s ) that originated i n the in t ima or migrated upwards from the m e d i a towards the in t ima l sur face . 1 2 7 Cy tok ines released from activated macrophages and endothelial cel ls further stimulate S M C migra t ion from the m e d i a into the i n t i m a . 1 4 9 However , at this early stage o f atherosclerotic development, S M C migra t ion is m i n i m a l due to interference from the support ing in t ima l extracellular matr ix ( E C M ) . The presence o f organized macrophage-derived foam cells and l i p i d car ry ing S M C s are def in ing characteristics o f a type II plaque. T y p e II plaques, also ca l led fatty streaks, (Figure 1.5) are the earliest s ign o f atherosclerosis v i s ib le to the human eye and have been studied for over a century. R u d o l f V i r c h o w , the father o f pathology, first described type II p l a q u e s 1 5 0 i n 'Endothdial Cell iDamaged Endothelial Cell 'Smooth Muscle Cell •Damaged SMC 'Red Blood Cell Monocyte Macrophage-Derived Foam Cell 0 Lipoproteins Reactive Oxygen Species Internal Elastic Lamina Figure 1.5. Type II fatty streak atherosclerotic plaque. U n d e r cont inued st imulus, monocytes migrate into the subendothelial region, become activated macrophages and accumulate o x i d i z e d L D L . The plaque progresses from a few focal areas o f l i p i d deposi t ion into layers o f macrophage-derived foam cel ls , and the occas ional l i p i d loaded S M C . 21 Chapter 1: Introduction children who had died of sickness or violent deaths establishing that cardiovascular disease may begin during infancy 15] The intermediate plaque or type III plaque is the third of the six A H A classifications (Figure 1.6). Small, extracellular lipid droplets are common throughout the plaque though a single large lipid core has yet to form. The small pools of extracellular lipid form among the migrating S M C s and macrophage-derived foam cells that disrupt the structural integrity o f the expanding intima and the underlying media . 1 2 7 Enzymes, such as matrix metalloproteinase (MMP)-2 and M M P - 9 , released from activated macrophages degrade collagen and other extracellular matrix elements responsible for the structural integrity of the vessel w a l l . 1 5 2 ' 1 5 3 Furthermore, matrilysin (MMP-7) and human metalloelastase (MMP-12) from macrophage-derived foam cells have been shown to have many non specific degradative actions against the E C M in atherosclerotic plaque caps that may result in separation of the cap region from the l ipid core. 1 5 4 A s the supporting matrix is degraded, the 'Endothelial Cell 'Damaged Endothelial Cell 'Smooth Muscle Cell (Damaged SMC q f l f t r SMC Derived Foam Cell 'Red Blood Cell Monocyte [Macrophage-Derived Foam Cell 0 Lipoproteins Reactive Oxygen Species Internal Elastic Lamina Figure 1.6. Type III atherosclerotic plaque. Small pools of extracellular l ipid form among macrophage-derived foam cells and migrating S M C s disrupting the structural integrity of the media and expanding intima. Very little luminal narrowing occurs at this stage. 22 C h a p t e r 1: I n t r o d u c t i o n 152 153 S M C s are r e l e a s e d f r o m t h e i r b a s a l l a m i n a e to f r e e l y m i g r a t e i n t o the i n t i m a l r e g i o n . ' A t h e r o s c l e r o t i c p l a q u e s w i t h e v i d e n c e o f S M C m i g r a t i o n a re c l a s s i f i e d as a t y p e I I I p l a q u e . T y p e I I I to t y p e I V a t h e r o s c l e r o t i c p l a q u e p r o g r e s s i o n i s t h e s tage w h e r e l u m i n a l n a r r o w i n g b e c o m e s i r r e v e r s i b l e . I f a n i n d i v i d u a l d e v e l o p s a t y p e I, II o r I I I a t h e r o s c l e r o t i c p l a q u e a n d t h e n a l t e r s t h e i r l i f e s t y l e , t a k e s u p r u n n i n g a n d q u i t s s m o k i n g f o r e x a m p l e , t h e t y p e I, II o r I I I a t h e r o s c l e r o t i c p l a q u e s c a n r e g r e s s a n d d i s a p p e a r . O n c e a p l a q u e h a s r e a c h e d s t age I V , i t b e g i n s to l o s e t h e a b i l i t y to r e g r e s s . C o m p o s i t i o n o f a t h e r o s c l e r o t i c p l a q u e s c a n c h a n g e f r o m l i p i d f i l l e d to a m o r e f i b r o u s o r g a n i z a t i o n ; h o w e v e r , t h e l u m i n a l n a r r o w i n g c h a r a c t e r i s t i c o f the t y p e I V , V a n d V I p l a q u e s i s p e r m a n e n t . T h e f i n a l t h r ee c l a s s i f i c a t i o n s : t y p e I V , t y p e V a n d t y p e V I p l a q u e s a re t h e a d v a n c e d t y p e s o f a t h e r o s c l e r o t i c p l a q u e s . T h e a d v a n c e d p l a q u e s a re a l l c h a r a c t e r i z e d b y t h e r e o r g a n i z a t i o n a n d a c c u m u l a t i o n s o f l i p i d s , c e l l s a n d e x t r a c e l l u l a r m a t r i x c o m p o n e n t s i n t h e i n t i m a r e s u l t i n g i n i n t i m a l t h i c k e n i n g a n d a r t e r i a l w a l l d e f o r m a t i o n i n c l u d i n g l u m i n a l n a r r o w i n g . 1 4 6 H o w e v e r , f u r t he r g e n e r a l c h a r a c t e r i z a t i o n o f t hese p l a q u e s i s c h a l l e n g i n g . I n the i n i t i a t i n g s tages o f a t h e r o s c l e r o s i s , the p a t t e r n o f p l a q u e d e v e l o p m e n t i s w e l l d e s c r i b e d a n d i s r e l a t i v e l y c o n s t a n t . P l a q u e c l a s s i f i c a t i o n s t y p e I - I I I r e p r e s e n t a d e v e l o p m e n t a l s e q u e n c e w i t h m i n i m a l v a r i a t i o n . A s p l a q u e s p r o g r e s s i n t o a d v a n c e d p l a q u e s t h e s u b s e q u e n t p r o g r e s s i o n b e c o m e s h i g h l y v a r i e d a n d a p l a q u e m a y a d v a n c e d i r e c t l y f r o m a t y p e I I I p l a q u e to a t y p e V , o r V I . 1 4 6 U n l i k e t y p e I I I p l a q u e s w i t h m a n y f o c a l l i p i d d e p o s i t s , t y p e I V p l a q u e s h a v e a c o n s p i c u o u s c e n t r a l a c c u m u l a t i o n o f e x t r a c e l l u l a r l i p i d w i t h i n the t h i c k e n e d i n t i m a , i n t e r c e l l u l a r l i p i d w i t h i n m a c r o p h a g e - d e r i v e d f o a m c e l l s , c o n s i d e r a b l e i n t i m a l d i s o r g a n i z a t i o n a n d a r t e r i a l t h i c k e n i n g ( F i g u r e 1.7).146 T h e s e p l a q u e s t y p i c a l l y d o n o t c a u s e a n a r r o w i n g o f t h e v e s s e l , b u t r a t he r c a u s e a n i n c r e a s e d o u t e r c i r c u m f e r e n c e o f t h e v e s s e l . T y p e I V p l a q u e s a re i n i t i a l l y e c c e n t r i c , t h o u g h as t h e y c o n t i n u e to p r o g r e s s t h e y m a y b e c o m e c o n c e n t r i c . 1 4 6 M i c r o s c o p y s h o w s tha t c a p i l l a r i e s b o r d e r o n t h e l i p i d c o r e , a n d m a c r o p h a g e s , as w e l l as l y m p h o c y t e s a re r e a d i l y f o u n d i n the a t h e r o m a ' s s h o u l d e r r e g i o n s . A b o v e the l i p i d l a y e r i s a r e l a t i v e l y n o r m a l i n t i m a c o n t a i n i n g a n a b u n d a n c e o f T - c e l l s a n d m a c r o p h a g e s . B e c a u s e o f t h e r e l a t i v e l y f l e x i b l e a n d w e a k n a t u r e o f the i n t i m a l r e g i o n i t i s p r o n e t o t h e d e v e l o p m e n t o f n a r r o w b u t d e e p tears o r f i s s u r e s . A c t i v a t e d m a c r o p h a g e s w i t h i n t h e p l a q u e p r o d u c e a n d r e l e a s e t i s s u e 23 Chapter 1: Introduction factor, a highly prothrombotic protein. 1 5 5 - 1 5 7 If a fissure does occur, and blood encounters tissue factor, blood clots w i l l immediately form 1 5 7 , 1 5 8 The type V plaques are characterized by a thickened, more fibrous plaque (Figure 1.8). Within the type V plaque classification there are several subgroups. The type V a plaques have evidence o f recent fibrous cap formation, typically characterized by S M C infiltration and extensive E C M deposition. The type V b is a plaque in which the lipid core and other regions of the plaque may be calcified. Finally, the type V c plaque is a plaque with minimal l ipid accumulation but a fibrous cap. A l l the type V plaques narrow the lumen of the vessel to varying degrees. 1 4 6 'Endothelial Ceil »Damaged Endothelial Cell 'Smooth Muscle Cell •Damaged SMC SMC Derived Foam Cell 'Red Blood Cell Monocyte ) Macrophage-Derived Foam Cell o Lipoproteins Reactive Oxygen Species Internal Elastic Lamina Fibrous Extracellular Matrix Figure 1.7. Type IV fibrous atherosclerotic plaque which is characterized by lipid accumulation in a central l ipid core, abundant population of macrophage-derived foam cells and lipid carrying S M C s . Like the type IV plaques, the type V plaques are prone to disruption and the subsequent development of thrombi (Figure 1.9). When these events (a disrupted surface, a hematoma, 2 4 Chapter 1: Introduction hemorrhage, or a thrombosis) do occur, the type I V or V plaques becomes a type V I plaque. T y p e V I plaques are c o m m o n l y referred to as c o m p l i c a t e d plaques. T h e d i s t i n g u i s h i n g feature is the presence o f a thrombus whether f r o m internal vessel rupture or external f ibrous cap A s s u r i n g . 1 4 6 T h e advanced plaques are the plaques o f concern because they are prone to disrupt ion and destabi l izat ion result ing i n t h r o m b o e m b o l i . T h r o m b i , part icular ly large t h r o m b i , superimposed o n the vessel w a l l can cause substantial n a r r o w i n g and even f u l l o c c l u s i o n o f the l u m e n . 1 1 8 'Endothelial Cell 'Damaged Endothelial Cell 'Smooth Muscle Cell •Damaged SMC SMC Derived Foam Cell 'Red Blood Cell Monocyte [Macrophage Derived Foam Cell o Lipoproteins Reactive Oxygen Species Internal Elastic Lamina Fibrous Extracellular Matrix Figure 1.8. Advanced type V fibrous atherosclerotic plaque. L i p i d a c c u m u l a t i o n i n the core, a f ibrous cap c o m p o s e d o f extracel lular m a t r i x a n d S M C s as w e l l as distinct organizat ion to the plaque are def in ing characteristics o f an advanced f ibrous atherosclerotic plaque. S o m e o f the S M C s i n the atherosclerotic plaque m a y have accumulated l i p i d . 1.4.7 The Danger of Atherosclerotic Plaques L u m i n a l n a r r o w i n g f r o m atherosclerotic plaques impedes b l o o d f l o w and alters b l o o d h e m o d y n a m i c s . Arter ies can become f u l l y o c c l u d e d b y atherosclerotic plaques and b l o o d f l o w w i l l be stopped leading to downstream tissue damage and potential ly death b y i s c h e m i a . 25 Chapter 1: Introduction However, thrombus formation as a consequence of atherosclerotic plaque erosion or rupture is thought to be the main cause of acute myocardial infarctions and of greater clinical Plaque erosion is the term describing the desquamation of the endothelium concern 159, 160 resulting in the exposure of the subendothelial E C M to circulating blood.1 6 1 Plaque rupture is the term used to describe a plaque that has torn open, exposing not only the surface of the plaque, but also the thrombogenic plaque core through deep fissures in the plaque.161' 1 6 2 Atherosclerotic plaque constituents, such as tissue factors and E C M elements, are highly reactive with blood resulting in rapid thrombus formation. 158, 163 'Endothelial Cell Damaged Endothelial Cell •Smooth Muscle Cell hDamaged SMC SMC Derived Foam Cell (Red Blood Cell Caft? Monocyte Ma crop ha ge- D erived Foam Cell o Lipoproteins Reactive Oxygen Species Internal Elastic Lamina Fibrous Extracellular Matrix Fibrin Deposits O Platelet Figure 1.9. Complicated advanced type VI atherosclerotic plaque. Plaque disruption with thrombus formation is a defining characteristic of a complicated atherosclerotic plaque. There are two primary types of thrombi that form in response to plaque rupture and erosion.118 The first is caused by haemorrhage into a fissure in the atherosclerotic plaque. This may cause rapid growth of the atherosclerotic plaque, even inducing further plaque rupture. The second type of thrombus is superimposed on the blood vessel wall and if large enough the lumen of the vessel may become fully occluded.118 Under the elevated pressure caused by the restricted blood flow, the entire, or a portion of the superimposed thrombi may 26 Chapter 1: Introduction break off the vessel wall and become lodged in a more distal, narrower arteriole or capillary. This w i l l lead to downstream full vessel occlusion and absence of blood flow to distal tissues resulting in ischemia. Ful l occlusion o f the vessel due to a thrombus leads to tissue damage and in severe cases tissue death. This response o f a thrombus formation as a consequence of plaque rupture is thought to be the major cause of myocardial and cerebral infarc t ions . 1 5 9 ' 1 6 4 1.4.8 Stable versus Unstable Atherosclerotic Plaques Not all plaques have the same likelihood of rupture . 1 6 1 ' 1 6 5 Based on the tendency to rupture, two basic phenotypes o f advanced atherosclerotic plaques are distinguishable: the stable phenotype and the unstable phenotype. 1 6 6 ' 1 6 7 Stable atherosclerotic plaques are characterized by an accumulation of l ipid within the core o f the plaque and the presence o f a thick, fibrous cap region between the endothelium and the deep lipid core. The l ipid component o f this plaque typically makes up less than 40% of the total plaque volume and the macrophage-derived foam cells are sequestered in the core region of the plaque with S M C s and E C M dominating the upper cap regions of the in t ima. 1 6 5 In contrast unstable atherosclerotic plaques characteristically have intracellular and extracellular l ipid deposits in excess of 40% of the total plaque composition. 1 6 5 A larger portion of the unstable plaque is composed of macrophage-derived foam cells spread through the entire thickness of the intima with a concomitant reduction in the number of S M C s . 1 6 2 ' 1 6 7 - 1 6 9 Table 1.3. Characteristics of stable and unstable atherosclerotic plaques - Ihick fibrous cap - The l ipid content < 40% - Macrophage-derived foam cells sequestered in core - S M C s dominating the upper regions of the atherosclerotic cap - Thin fibrous cap - The l ipid content > 40% - Increase in macrophage-derived foam cells throughout entire plaque thickness - Reduction in S M C s Plaque phenotypes are not permanent; there exists the possibility o f shifting between the stable and unstable phenotypes in response to appropriate s t imul i . 1 6 1 ' 1 6 5 If the stimulus includes the main risk factors for atherosclerosis including high serum cholesterol, hypertension and/or a chronic inflammatory response, then L D L particles w i l l continue to 27 Chapter 1: Introduction accumulate in the intimal region and be oxidized by the R O S species in the plaque tissues. Oxidized L D L w i l l lead to further macrophage infiltration and activation, further cytokine production by the macrophage-derived foam cells and degradation of the E C M components 162 167 of the plaque; all processes leading to an unstable atherosclerotic plaque phenotype. ' B y contrast, i f the offending stimulus is removed, for example a patient's serum cholesterol and hypertension are brought under control and the inflammatory response resolves then the atherosclerotic plaque w i l l begin to shift towards the stable fibrous phenotype. Invading macrophages w i l l be able to remove debris, and potentially aid in the removal of L D L from the plaque, thereby preventing further tissue damage. 1 7 0 ' 1 7 1 S M C s that have migrated into the cap region of the plaque w i l l produce E C M elements to stabilize the plaque and sequester the l ipid in the atherosclerotic plaque c o r e . 1 6 7 ' 1 7 2 The luminal narrowing of the plaque w i l l not recede; however, the composition of the plaque w i l l change resulting in a more stable, fibrous plaque. Stable atherosclerotic plaques have much less tendency to rupture or suffer from endothelial erosion. 1 6 7 The predisposition towards unstable atherosclerotic plaques is correlated with the main risk factors for atherosclerosis. Activities such as smoking, a high fat diet and a sedentary lifestyle predispose an individual for the physiological conditions that w i l l push existing atherosclerotic plaques towards the unstable phenotype. Wi th the global population experiencing rising levels of all major risk factors, it is not surprising that cardiovascular related mortalities continue to rise. What are not fully understood are the mechanisms by which air pollution contributes to the observed increase in mortality. Epidemiological studies repeatedly put forward robust positive associations between cardiovascular morbidity and mortality and particulate matter air pollution yet the underlying biological mechanisms still remain unidentified. 1.5 Proposed Mechanisms for Particulate Matter Mediated Cardiovascular Events The scientific community is currently considering three primary mechanisms by which particulate matter air pollution may bring about cardiovascular events. These are the inflammatory mechanism, the dysfunction of the autonomic nervous system mechanism and the cardiac malfunction mechanism. 5 4 28 Chapter 1: Introduction 1.5.1 Inflammatory Mechanism A s the inhaled particulates interact with the alveolar macrophages a systemic inflammatory response is initiated. PMio induced inflammation may contribute to the chronic inflammatory nature of atherogenesis.5 4 A i r pollution interacts with the body at the air-tissue interface in the lungs. Contained below the surfactant coating o f the alveoli are the alveolar macrophages. The primary function o f the alveolar macrophage as a professional phagocyte is to protect the lungs from inhaled particulate material, including both airborne particulates and pathogens 4 0 In cases where the alveolar macrophages becomes overburdened with inhaled particulates, protective responses such as cytokine secretion and initiation of an inflammatory response may become damaging to the lung tissue. 1 7 4 Previous research has shown that in response to low levels of PMio exposure, there is an elevation in cytokine production by alveolar macrophages, specifically T N F - a , IL-6, and G M - C S F . 1 0 4 ' 1 7 5 In cases of sepsis induced by bacterial lipopolysaccharides, these cytokines are able to enter the circulation 1 7 6 thereby inducing the systemic inflammatory response. Though it has not been confirmed in vivo, a similar process is believed to occur following PMio exposure. When alveolar macrophages are exposed to PMio in vitro, increased levels of T N F - a were found in the culture media . 1 0 4 Furthermore, Mukae and colleagues 1 0 4 have shown that when the supernatants from these experiments are instilled into the lungs of rabbits, a systemic immune response occurs characterized by increased circulating polymorphonuclear leukocyte and band cell counts. Further experiments revealed monocytes had a decreased transit time through the bone marrow and were released more rapidly in response to the P M i o stimulus. 1 0 5 These experiments confirm that alveolar macrophages release mediators following phagocytosis o f PMio which can induce a systemic inflammatory response and cause the stimulation o f the bone m a r r o w . 1 0 4 ' 1 0 5 Certain cytokines, in particular T N F - a , are believed to play a pivotal role in systemic immune stimulation. Driscoll and colleagues 1 7 7 have suggested that the mechanism of T N F - a involvement in the inflammatory response is via an indirect network of cell to cell cytokine interactions. For example, in response to T N F - a secretion from alveolar macrophages, epithelial cells and other alveolar macrophages are stimulated to release IL-8, IL-1 , and 29 Chapter 1: Introduction M C P - 1 . 1 3 7 ' 1 7 7 Increased epithelial permeability allows these cytokines to enter circulating blood, while IL-8, a strong neutrophil chemoattractant, draws neutrophils into the lung interstium and air-space. 1 7 7 The neutrophils and macrophages phagocytize the PMio particles in an attempt to remove the offending agent, but they themselves become activated and continue to release cytokines that prolong the cellular infiltration. The IL-1 and T N F - a that have entered the systemic circulation play a role in bone marrow stimulation. Work from our laboratory has confirmed an increase in circulating band cells (immature neutrophils) and polymorphonuclear leukocytes ( P M N ) in blood in response to PMio exposure. 1 7 8 This effect is believed to be mediated by the T N F - a released from alveolar macrophages 1 0 4 ' 1 7 9 activated through processing PMio deposited deep in the lungs. Systemic immune activation results in a shortening of the transit times of P M N s through the mitotic and post-mitotic bone marrow pools . 1 0 6 ' 1 7 8 The increased numbers o f circulating P M N s ordinarily would aid in the elimination o f infectious agents. However, Friedman and 180 colleagues have shown that an elevation in the peripheral blood leukocyte count has been associated with an increased mortality. The nature of this link is not known, nor is it known i f increased mortality is caused by elevated levels o f a single leukocyte type, or a combination o f several leukocyte types. Resolving these questions may be important in understanding the clinical relevance of elevated levels o f circulating P M N s following PMio exposure. Further evidence of acute immune stimulation during periods of elevated particulate air pollution is increased circulating levels of the liver protein C-reactive protein ( C R P ) . 1 8 1 C R P is a protein involved in the induction of the complement immune system. In response to cytokine regulation, circulating levels of C R P can increase 1000-fold during an acute immune response. 1 8 1 Alveolar macrophages are known to secrete C R P inducing cytokines 1 3 7 , 179 which may account for the elevated circulating levels of C R P following PMio exposure and it has been demonstrated in the endothelial cells, macrophages and S M C s of atherosclerotic p laques . 1 8 3 ' 1 8 4 Furthermore, due to its rapid response rate and short half life, C R P is emerging as a powerful predictor of cardiovascular r i s k . 1 8 5 , 1 8 6 The physiological role o f C R P as a component of the 30 Chapter 1: Introduction innate immune system is well characterized. It binds to phospholipids of damaged cells 188 enhancing scavenger recognition and phagocytosis by macrophages. O f particular significance to cardiovascular events, C R P activates macrophages to secrete cytokines and tissue factor while priming them for L D L uptake. C R P also affects endothelial cells by inducing their expression o f adhesion molecules, secretion of IL-6 and endothelin-1, and decreasing the synthesis and availability of nitric oxide synthase. 1 9 0 The observation of elevated C R P following PMio exposure is further indication of the consequences o f PMio exposure on the cardiovascular system. The elevation of the peripheral blood leukocyte count, secretion of cytokines from alveolar macrophages and extensive effects o f C R P are all features of a pro-inflammatory, pro-atherogenic environment. For a patient who has severe atherosclerosis, this combination may be enough to initiate the events that further destabilize an advanced unstable atherosclerotic plaque resulting in a myocardial infarction, a cerebral infarction, and potentially death. 1.5.2 Dysfunction of the Autonomic Nervous System There is mounting evidence of changes to the autonomic nervous system following exposure to particulate matter air pollution. A n early study reported that the risk of arriving dead at the hospital was significantly higher on high air pollution days, 1 1 thus incriminating sudden deaths caused by arrhythmias and myocardial infarctions as the predominate cause o f the increased mortality. 1 9 1 A 1999 study by Liao and colleagues 1 9 2 was the first to examine the effects of particulate matter on heart rate variability. Subsequent studies have confirmed decreased heart rate variability and increased blood pressure in at risk populations such as the elderly or those with pre-existing condit ions 1 9 3 " 1 9 7 as well as in young healthy individuals. 1 9 8" 2 0 0 However, the underlying mechanisms o f autonomic dysfunction in relation to particulate matter exposure are not understood. There is speculation in the literature that the effects on heart rate variability and blood pressure are mediated directly from the lungs or directly from the heart. Within the lungs the response would occur via pulmonary reflexes or a pulmonary inflammatory response. Within the myocardium, the potential effects of particulate matter on ion channels, on the ischemic 31 Chapter 1: Introduction response of the myocardium, and on the potential for inflammatory responses leading to 201 endothelial dysfunction have all been suggested as possible mechanisms. There is a growing body of evidence to support both the heart and the lung hypotheses. Studies by Seaton and colleagues 2 0 1 suggest direct stimulus via pulmonary afferent fibres from the lungs to the autonomic nervous system might contribute to immediate changes in heart rate variability caused by particulate matter. A study by Park and colleagues 1 9 5 found that the effect of particulate matter on heart rate variability was diminished in patients taking calcium channel blockers thereby suggesting the involvement of the sympathetic nervous system. Magari and colleagues concluded that changes in heart rate variability in middle aged healthy men were mediated by some aspect of autonomic function that had been compromised as a consequence o f air pollution exposure. However, the authors were unable to determine whether the effects were mediated by increased sympathetic or decreased vagal activity. Thus the literature provides evidence that particulate matter exposure is affecting heart rate, heart rhythm and heart regulation. The precise nature of the mechanisms involved remains unknown, as does the extent of consequences of changes in autonomic regulation of the heart following particulate matter exposure. 1.5.3 Cardiac Malfunction This final proposed mechanism involves particulate induced cardiac malfunction caused by particulate translocation into the blood s t r e a m 4 6 ' 4 8 ' 5 0 leading to local damage to the heart muscle 1 9 5 as well as the l iver 4 8 and the vasculature. 5 4 O f the three proposed mechanisms, the cardiac malfunction mechanism has the least research and supporting data. The concept o f PMio induced cardiac malfunction is a field that requires more research and understanding to determine i f in fact particles are able to translocate into the body and cause direct damage to the myocardium. In spite o f all these hypotheses and data, the mechanism whereby air pollution exposure initiates cardiovascular morbidity and mortality remains largely unknown. A s discussed above, measurable physiological changes in blood flow, blood pressure, and heart function have been documented, yet the observed changes are relatively small. What is not small is the number of deaths annually that are attributed to PMio exposure. In order to minimize air 32 Chapter 1: Introduction pollution related morbidity and mortality, the mechanisms linking respired particulates to myocardial infarctions, cerebral infarctions, and sudden death must be elucidated. 1.6 Hypothesis The field of PMio toxicity is growing and providing greater insight into the positive association between cardiovascular related morbidity and mortality. Epidemiological evidence is the most extensive, supported by preliminary chemical studies investigating particle size, composition and deposition as well as preliminary mechanistic studies looking at measurable variables in human populations such as heart rate variability, blood pressure changes, and chronic inflammation. The field that remains relatively unexplored is that of physiological and pathological changes resulting from PMio exposure. What is the sequence of events that begins with the deposition o f particulates in the lungs, and leads to the final endpoint of an acute cardiovascular event? The global prevalence o f atherosclerosis is very high and an understanding of morphological alterations in atherosclerotic plaques as a consequence of PMio exposure would enable improved prevention in all populations and improved treatment of at risk populations. We hypothesize that with PMio exposure ultrastructural changes occur in the cap regions of atherosclerotic plaques that compromise endothelial stability, integrity and cap composition. The overarching objective of this series of studies was to examine atherosclerotic plaques from W H H L rabbits exposed to PMio for morphological features o f remodelling and reorganization, with particular emphasis placed on the integrity o f endothelium and its underlying extracellular matrix, the cell population subtending the endothelium and evidence of cap degradation. 33 Chapter 1: Introduction 1.6.1 Specific Aims The specific aims of each study presented in this dissertation were: 1. To compare the fine organization o f atherosclerotic plaques from PMio exposed rabbits to the organization of atherosclerotic plaques from control rabbits for changes indicative of a shift to a more vulnerable atherosclerotic plaque phenotype. 2. To define the organization of the E C M upon which the plaque endothelium sits in the control W H H L rabbits and compare that with the organization o f the E C M in PMio exposed W H H L rabbits where macrophage-derived foam cells have accumulated in the subendothelial region. 3. To quantitatively assess differences in the area o f endothelial cell / dense extracellular matrix contacts in atherosclerotic plaque caps of PMio exposed and control W H H L rabbits. 4. To determine whether the subendothelial reticulum o f dense E C M of atherosclerotic caps is histochemically similar to the known composition o f basal lamina of normal artery and therefore able to perform similar functions. 5. To define any observed differences in endothelial topography and document leukocyte trafficking on plaque surfaces of PMio exposed and control W H H L rabbits. 34 CHAPTER 2 General Methodology 2.1 Animals Protocols in this study were approved by the Animal Experimentation Committee of the University o f British Columbia (see Appendix II for certificate) and involved 16 female Watanabe heritable hyperlipidemic ( W H H L ) rabbits (Covance Research Products Inc., Denver, P A ) weighing 3.5 ± 0.4 kg (mean ± SD). A l l rabbits were fed standard rabbit chow throughout the study and were sacrificed between 42 and 46 weeks of age. W H H L rabbits have a spontaneous 4 amino acid deletion in the cysteine-rich ligand binding domain of the L D L receptor gene, the same gene associated with human familial hypercholesterolemia. 2 0 2" 2 0 4 In both humans and rabbits, this genetic defect produces a drastic reduction of plasma membrane L D L receptors on endothelial cells and leads to the decreased clearance of lipoproteins, resulting in extremely high levels o f L D L in the circulating blood. Z U J ' ^ Humans can have many possible mutations and can be heterozygotes or homozygotes for these mutations. 2 0 4 One person in every 500 people is a heterozygote resulting in half the normal level of L D L receptors being produced by their cells. Heterozygotic patients suffer from both a decreased clearance o f L D L from their plasma and an increased L D L production resulting in plasma L D L levels twofold to threefold higher than plasma L D L levels in healthy people. Routinely symptomatic vascular disease develops in the 4 t h or 5 t h decade of l i f e . 2 0 4 Homozygotic patients with two defective genes have extremely low levels o f expressed L D L receptor and a resulting sixfold to eightfold increase in plasma L D L levels manifesting as early as 20 weeks in embryonic development. These patients typically develop symptomatic vascular disease in their 2 n d decade of l i f e , 2 0 4 as well as skin xanthomas and l ipid deposits in most tissues. 2 0 3 Genetic and biochemical studies show 35 Chapter 2: General Methodology conclusively that the disease is caused by a mutation in the L D L receptor gene resulting in a defective gene. The W H H L rabbit is a model that has been systematically studied since 1973 when a Japanese veterinarian made the observation that one of his male New Zealand white rabbits had elevated plasma L D L levels . 2 0 7 After breeding and selecting for rabbits with elevated levels of plasma L D L , Yoshio Watanabe established the W H H L rabbit model on the New Zealand white rabbit background. Just as with human familial hypercholesterolemia, breeding the W H H L rabbit produced heterozygotes or homozygotes for the gene mutation. 2 0 4 Atherosclerosis develops without the need for cholesterol feeding in the homozygotic rabbits by five months of age and progresses in both severity and extent of disease as the animal ages. 2 0 5 The W H H L homozygotic rabbit model differs from human familial hypercholesterolemia homozygotes in that the rabbits have hypercholesterolemia and hypertriglyceridemia whereas humans only have hypercholesterolemia with normal triglyceride levels . 2 0 4 The explanation for this is very simple, rabbits have elevated baseline triglyceride levels when compared to humans and remain so with or without the L D L receptor deficiency 2 0 8 When compared to atherosclerotic animal models that require cholesterol feeding for atherosclerotic plaque development, the W H H L homozygotic rabbit model develops atherosclerotic plaques on a normal diet and the plaques are indistinguishable from human atherosclerotic plaque thus making this model ideal to study the etiology of atherosclerosis. 2.2 Urban Particulate Matter The urban particulate matter (EHC-93) used in this study was provided through the courtesy of Dr. R. Vincent at Health Canada. The particulates were collected throughout all seasons from the troposphere at Tunney's Pasture in Ottawa, Canada in 1993 using a single - pass, videlon bag house filtration system located at the Environmental Health Centre. 100% of the intake air was ambient air and there was no recirculation of indoor air. The sample was vacuumed off the air filter and mechanically sieved through multiple mesh filters, the last of which was a nytex mess monofilament filter with a 36 um pore s ize . 2 0 9 N o compression or l iquid factors were used to push the particles through the filter; rather the sieves were shaken 36 Chapter 2: General Methodology-back and forth for days to allow gravity to pull the sample through. 2 0 9 Care was taken to not alter the size, composition or toxicity o f the collected sample. 2 0 9 Analysis of EHC-93 showed a mean diameter of 0.8 ± 0.4 u m (mean ± SD), with 99% of the number of particles < 3.0 p m . 2 1 0 EHC-93 particles resuspended in water and then dried down on a silica wafer prior to sputter coating with 8 nm of a gold palladium alloy were viewed using a Hitachi S4700 Field Emission Gun S E M . Particles had variable shapes and sizes. In agreement with the size Figure 2.1. EHC-93 particulate matter, a) Particulates varied in size with a few coarse particles smaller than 10 p m (*), several fine particles between 2.5 and 0.1 pm (arrow) and many ultrafine particles smaller than 0.1 p m (arrowhead), b) Pollen grains were observed in the EHC-93 sample, c) Particles smaller than 2.5 pm exhibited variations in shape and size. 37 Chapter 2: General Methodology analysis, the number of particles observed appeared to be approximately inversely proportional to the particle size (Figure 2.1). Repeated observations were made of pollen grains coated with fine and ultrafine particulates. The composition of these particles has been fully characterized as shown in Table 2.1. Particulates collected from urban centers differ in composition based on the industry within the region, the proximity to the ocean, and weather patterns. 2 4 ' 5 1 ' 5 8 Therefore, EHC-93 has a unique composition from other urban collected particulates. When compared to eight other 910 urban particulates, the EHC-93 sample had high levels of A l , C a and Na . In terms o f size, 9 1 0 the EHC-93 sample is similar to other urban particulate samples. Size is an important determinant of particulate deposition in the lungs and physiological effects of exposure. 3 7 ' 4 0 Table 2.1. The composition of EHC-93 urban particulate matter. Elemental Contents of Acidity, Sulphate, and Ammonia of E H C - 9 3 2 1 0 EHC-93 (pg/g)210 Aluminium 9.8 mg/g H + 0 Arsenic - S O " 4 4.45 x 10 4 Barium 295 pg/g N H 4 104 Boron 81.2 pg/g Polycyclic Aromatic Hydrocarbons of Cadmium 7-3 pg/g EHC-93 (pg/g)210 Calcium 109 mg/g Acenaphtene 0.20 Chromium 42.3 pg/g Anthracene 0.54 Cobalt 5.0 pg/g Benzo [a] anthracene 1.10 Copper 845 pg/g Benzo [b] fluoranthene 2.78 Iron 14.9 mg/g Benzo[k]fluoranthene bdl* Lead 6.7 mg/g Benzo[a & bjfluorene 0.23 Magnesium 7.2 mg/g Benzo[ghi]perylene 1.52 Manganese 483 pg/g Benzo[a]pyrene 0.95 Molybdenum 4.6 pg/g Benzo[e]pyrene 1.09 Nickel 69.6 pg/g Chrysene 1.66 Sodium 20.6 mg/g Dibenz[a,c & a,h] anthracene bdl Strontium 272 pg/g Fluoranthene 2.47 T in 1.2 mg/g Indeno[l,2,3-cd]pyrene 1.19 Titanium 929 pg/g Perylene 0.28 Vanadium 90.4 pg/g Phenanthrene 1.83 Zinc 10.4 mg/g Pyrene 2.11 *bdl =below detectable levels 38 Chapter 2: General Methodology 2.3 PMio Exposure 2.3.1 Exposure Protocol Five mg of PMio suspended in 1 m l of sterile saline was instilled into the upper airway of PMio exposed W H H L rabbits (n = 8) and normal saline was delivered in the same manner to the control W H H L rabbits (n = 8) as previously described in the l i t e r a tu re . 1 0 5 ' 1 0 6 ' 1 7 8 The rabbits were 38-42 weeks of age at the beginning of the exposure protocol. The exposure protocol was as follows: for each exposure, five mg of particles were weighed out, added to 1 ml of sterile saline and the solution was sonicated for several minutes. The rabbits were anaesthetized with 5% halothane (Bimedia /MTC Animal Health Inc, Cambridge ON) and the experimental suspension of PMio (EHC-93), or 1 m l of saline was deposited just above the larynx using a blunt curved needle. The needle was removed and the W H H L rabbit was rolled from side to side several times to ensure a more even dispersion of the particles throughout the lungs. The rabbits were monitored as they awoke from the halothane and then placed back in their cages and monitored after exposure. This protocol was performed twice per week for four weeks. The rabbits were sacrificed with an overdose of sodium pentobarbital (Bimedia /MTC Animal Health Inc, Cambridge ON) when the exposure protocol was complete and tissues were sampled. 2.3.2 Exposure Level The exposure concentration was 5 mg of P M i o suspended in 1 m l of sterile saline which is a particle dosage calculated to be relevant to human exposure by Mukae and colleagues. 1 7 8 That study documented that only 20% of the 5 mg of PMio deposited just above the larynx in the rabbit is aspirated into the lungs and only 4% reached the alveolar surface. 1 7 8 Suwa and colleagues {Suwa, 2002 #114} determined that a dosage of 5 mg o f PMio instilled in this way twice per week for four weeks, resulted in an alveolar surface concentration of 2.9 ng/cm 2 per installation of PMio or an alveolar exposure of 23.2 ng/cm 2 over the entire 4 week experimental period. A s these values are comparable to human exposure of 150 pg/m 3 calculated for a natural exposure that occurred over 20 days 1 7 8 or similar to human exposure 39 Chapter 2: General Methodology during the South East As i a forest fires of 1997 9 5 we think that the exposures achieved in our experiments are reasonable and comparable to naturally occurring human exposure. 2.4 Tissue Fixation, Excision and Processing for Electron Microscopy Two different protocols were used to fix the aortas in situ (Figure 2.2). In the first protocol, W H H L rabbits (n = 3 control, n = 3 PMio exposed) were sacrificed with an overdose of sodium pentobarbital and the abdominal aorta of the rabbit was rapidly exposed. The aorta was fixed in situ by adding 4% glutaraldehyde ( B D H Inc, Toronto, ON) in 0.1 M cacodylate buffer (Canemco, St Laurent, QC) (pH 7.4) into the abdominal cavity of the rabbit while the heart was still beating. Prior to the addition of fixative, the abdominal aorta was hemostated at the iliac bifurcation and then immediately below the diaphragm. The fixative was left in the abdominal cavity for ten minutes. The abdominal aorta was then excised and further immersion fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer. This protocol allowed fixation of blood elements in the vessels at physiological intravascular pressures. Processing for transmission electron microscopy ( T E M ) was continued following fixation. In order to be able to view the blood vessel lumen by scanning electron microscopy (SEM) , the blood must be flushed out prior to fixation which necessitated a second protocol. A s before, W H H L rabbits (n = 5 control, n - 5 PMio exposed) were sacrificed with an overdose of sodium pentobarbital. However, in this protocol the aorta was perfused with heparinized Krebs buffer until the exiting fluid began to lighten in colour. The aorta was then perfusion fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer at 100 cm of water pressure for ten minutes. Perfusion solutions were infused retrograde through the right carotid artery, down the aorta and then drained from the inferior vena cava. The end result was perfusion of the aorta in the direction of blood flow. The entire aorta from the heart to the iliac branch was carefully excised and further immersion fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer as the first step of processing for electron microscopy. N o differences in the quality o f fixation were observed between these two protocols. A l l excised samples for electron microscopy were immersion fixed for 90 minutes in 2% glutaraldehyde in 0.1 M cacodylate buffer in vitro and rinsed with 0.1 M cacodylate buffer. 40 Chapter 2: General Methodology Figure 2.2. Tissue processing protocols for transmission and scanning electron microscopy studies. The blood vessel was then cut longitudinally into large pieces, roughly 1 cm in length. Longitudinal halves were then randomly divided into S E M samples and T E M samples, with care to ensure the presence o f atherosclerotic plaques for both S E M and T E M analysis. T E M samples were cut into small pieces, between 1 and 3 m m 3 in size. S E M samples were kept as large as possible, restricted only by the size of the aluminium stub used for S E M viewing. A l l samples were then fixed for 1 hour in a solution of 1% Os04(Marivac Inc, Montreal, QC) , and 1% potassium ferrocyanide (K4[Fe(CN)6]) (Fisher Chemicals, Fair Lawn NJ) in 0.1 M cacodylate buffer. Samples were then rinsed three times with distilled water prior to dehydration through a graded acetone series (30%, 50%, 70%, 90%, 100%, 100%, and 100%). Following the graded dehydration series, samples destined for S E M studies were chemically dried using hexamethyldisilazane (Sigma-Aldrich, St Louis, M O ) in a Biowave Microwave 41 Chapter 2: General Methodology (Pelco International, Redding, C A ) . After mounting specimens on aluminium stubs using adhesive spots, all samples were sputter coated with a gold / palladium alloy in a Nanotech SEM-Prep II Sputter Coater (Nanotech, Manchester, U K ) . Samples were viewed in a Hitachi S4700 Field Emission Gun S E M (Hitachi, Rexdale, ON) . For Light Microscopy ( L M ) and T E M , samples were infiltrated with epon 812 resin (Electron Microscopy Sciences, Hatfield, P A ) using a gradual infiltration protocol. Tissues were then oriented in a mold filled with 100% freshly made epon 812 resin and polymerized overnight at 65°C. Blocks were trimmed and 0.5 pm thick sections were cut using a Leica E M U C 6 Ultramicrotome (Leica Microsystems, Richmond H i l l , ON) . The L M sections cut from these blocks were mounted on glass slides, and stained with toluidine blue O (TBO) for 10-20 seconds, cover slipped and examined with a N ikon Labophot-2 Light Microscope (Nikon, Mississauga, ON) . The T E M thin sections were cut at 60-80 nm, picked up on formvarr coated slot grids and stained for 4 minutes each with uranyl acetate and lead citrate. A l l sections were examined using a Tecnai 12 T E M (FEI Company, Hillsboro, OR) . 2.4.1 Inclusion Criteria for Tissue Analysis The objective of this study was to document architectural changes in pre-existing atherosclerotic fibrous cap ultrastructure observed following PMio exposure. Tissue samples were chosen to best address that objective. Only tissues that met the following inclusion criteria were analyzed: plaque thickness o f greater than 100 pm; core regions of a plaque; or i f a shoulder was present, sampling began 250 pm (measured along the internal elastic lamina) from the shoulder. Plaques thicker that 100 pm were analyzed to ensure that advanced, existing atherosclerotic plaques were the subject of observations. Shoulders were avoided because by nature they are regions of plaque expansion which are characteristically unstable, missing elements of stability such as a fibrous cap populated by smooth muscle 168 cells. We confined our morphometric analysis to the normally more stable fibrous cap region over the cores of the atherosclerotic plaques. Separate methodologies were used to address specific areas of interest. Details are described in the appropriate chapters. 42 Chapter 2: General Methodology 2.5 Tissue Fixation, Excision and Processing for Histochemistry and Immunohistochemistry The animals used in this specific study were not the same animals studied in other studies described within this dissertation. However, these animals were exposed in the same fashion and at the same time as the rabbits used in other studies in this dissertation. Following the exposure protocol, the W H H L rabbits were sacrificed with an overdose of sodium pentobarbital. The aortas were rapidly excised, opened longitudinally and digitally photographed (for another experiment 1 0 6). The aorta was then sectioned and oriented in a cassette prior to fixation in 10% neutral buffered formalin. The tissue was paraffin embedded by technicians in the histology facilities at the i C A P T U R E Centre. The blocks have been stored at room temperature until borrowed for the investigations described herein. Further details on sectioning and immunohistochemical staining protocols are provided and discussed in Chapter 5. 43 CHAPTER 3 Alterations in Atherosclerotic Plaque Organization 3.1 Introduction Atherosclerosis is a chronic disease that takes decades to become clinically relevant. Although it has been described as a series o f steps, atherosclerosis is a long continuum of a 1 C Q O i l 0 1 0 slowly progressing chronic disease. Pioneering work done by Michael Davis ' ' and Erl ing F a l k 1 1 2 ' 2 1 3 ' 2 1 4 established that there are two atherosclerotic plaque phenotypes: stable and unstable atherosclerotic plaques. The stable atherosclerotic plaque is characterized by a fibrous cap of S M C s and E C M that isolates extracellular lipids, macrophage-derived foam cells and cell debris deep in the core of the plaque away from the blood vessel lumen. The l ipid volume is less than 40% of the total plaque volume and there is a conspicuous S M C population. 1 6 5 Clinical ly these plaques are associated with a very low risk of rupture and thromboembolic complications. 2 1 5 In contrast, vulnerable plaques are organized in many different ways which makes clinical intervention challenging. They are typically characterized by a large gruel core made up of extracellular l ipid and cell debris that are in excess of 40% of the plaque v o l u m e . 1 6 5 ' 1 7 3 , 2 1 6 This extensive volume of l ipid deposition may be covered by a thin fibrous cap, containing a reduced collagen content and S M C population as well as an increased accumulation of macrophages when compared to stable atherosclerotic p l a q u e s . 1 6 8 ' 2 1 5 ' 2 1 7 Vulnerable plaques are prone to rupture and thrombus formation, making them much more clinically relevant due to the elevated risk o f thromboembolic events. 1 6 2 ' 2 1 5 ' 2 1 6 O f particular importance, 44 Chapter 3: Alterations in Atherosclerotic Plaque Organization examination of ruptured atherosclerotic plaques in patients with an acute myocardial infarction revealed that inflammation by monocytes was a crucial determinate o f location of 9 1 7 99(1 plaque rupture and potentially endothelial erosion. A i r pollution exposure is positively associated with cardiovascular morbidity and mortality. Three potential mechanisms have been proposed to contribute to this outcome: the inflammatory mechanism, the dysfunction of the autonomic nervous system mechanism and the cardiac malfunction mechanism (refer to Chapter 1, section 1.5 for full descriptions). 5 4 The experimental emphasis of this thesis is focused on the inflammatory mechanism. Evidence in the form of increased C R P levels 2 2 1 and increased circulating band ce l l s 1 0 6 are indicative of a systemic inflammatory response occurring following PMio exposure. 2 2 2 Using O i l Red-0 staining Suwa and colleages 1 0 6 described increased total l ipid accumulation in atherosclerotic plaques from W H H L rabbits following exposure to PMio (Table 3.1). Furthermore, overall plaque size was larger and there was evidence o f increased inflammatory cell infiltration in atherosclerotic plaques following PMio exposure. 1 0 6 In a 107 separate study by Sun and colleagues, increased levels o f l ipid were described in plaques from A p o E -/- mice fed high fat chow and exposed to low levels o f PM2.5when compared to mice on high fat chow but without PM2.5 exposure (Table 3.2). 1 0 7 Additionally, an increased population of macrophages in the intima and media of atherosclerotic plaques were documented following PM2.5 exposure for mice on high fat chow. These observed changes are consistent with a transformation to the unstable or vulnerable atherosclerotic plaque Table 3.1. Major findings from Suwa and colleagues' exposure of W H H L rabbits to PMio twice per week for four weeks. t foam cell population Movat Movat O i l red O Movat B r d U B r d U t extracellular l ipid deposition t total l ipid deposition t extracellular matrix deposition t circulating band cells and P M N s t cellularity I advanced atherosclerotic plaques Light microscopy Light microscopy Trend toward more atherosclerotic plaque 45 Chapter 3: Alterations in Atherosclerotic Plaque Organization Table 3.2. Major findings from Sun and colleagues' exposure of ApoE -/- mice to PM2.5 for six hours a day for five days per week for six months. : i::'(;t0i'_fLUSt.V>-. ,:5.-: •:-Li})'ivJ'"' 1—ra 'X lEV ' t atherosclerosis plaque size M R I imaging t macrophage population in media and intima C D 68 t l ipid deposition O i l Red-0 t inducible N O S and 3-Nitrotyrosine i N O S - endothelial N O S eNOS | vasoconstriction responses Functional studies phenotype. These studies used light microscopy, M R I imaging and immunohistochemistry to provide an overview of changes occurring as a consequence of air pollution exposure. Light microscopy is a powerful tool to image large areas, yet it is unable to resolve cell borders, location of l ipid deposition (intracellular versus extracellular), and definitive aspects of cell to cell and cell to E C M contacts and interactions that may contribute to plaque instability. In this study, the focus was to document ultrastructural changes occurring within atherosclerotic plaques as a consequence of PMio exposure. The resolving power of electron microscopy was used in subsequent chapters to investigate changes in cell organization, cell to cell and cell to E C M interactions, E C M organization and cap composition. However, prior to high resolution, small sample area studies, a light microscopic investigation o f tissues fixed for electron microscopy was done to determine overall structural changes in the organization of atherosclerotic plaques from W H H L rabbits exposed to PMio compared to plaques from unexposed rabbits. In this chapter, we used tissue preserved for electron microscopic studies by additive fixation with glutaraldehyde and osmium tetroxide that enabled us to better recognize cell types, lipids and E C M elements. Based on what is known o f the unstable atherosclerotic plaque phenotype and the characteristics o f plaque rupture, we would predict that repeated exposure to PMio leads to increased l ipid deposition, decreased S M C s in the plaque fibrous cap, increased inflammatory cells throughout the plaque, areas of monocyte infiltration and destabilization of the endothelium. 46 Chapter 3: Alterations in Atherosclerotic Plaque Organization 3.2 Aim To compare the fine organization of atherosclerotic plaques from PMio exposed rabbits to the organization of atherosclerotic plaques from control rabbits for changes indicative of a shift to a more vulnerable atherosclerotic plaque phenotype. 3.3 Methodology Exposure protocols and tissue processing protocols were outlined in Chapter 2. The epon embedded blocks destined for T E M analysis were used as follows for this investigation. 3.3.1 Qualitative Protocol 0.5 pm sections were cut from all 653 epon embedded blocks of tissue from abdominal aorta samples obtained from the 16 W H H L rabbits. Eight o f these rabbits had been exposed to PMio , the remaining eight rabbits were controls. The rabbits exhibited variable amounts o f plaque formation in their abdominal aorta, and following evaluation of a 0.5 pm section cut from each of the 653 epon embedded blocks, 88 embedded blocks of tissue containing atherosclerotic plaques from 7 control rabbits and 74 embedded blocks o f tissue containing atherosclerotic plaques from 5 PMio exposed rabbits were retained in the study. Table 3.3 outlines the distribution of abdominal aorta samples from each W H H L rabbit. Rabbits with zero usable samples were completely removed from the study. Using light microscopy, the plaque morphology of each sample of abdominal aorta was characterized and documented. The core region of the atherosclerotic plaques was o f more interest than the shoulder regions and particular emphasis was placed on the upper fibrous cap region and endothelium. 3.3.2 Quantitative Protocol Inclusion criteria for quantification o f the macrophage-derived foam cell population subtending the endothelium were: 1. plaque thickness must be greater than 100 pm; and 2. only non-shoulder regions of a plaque, or, i f a shoulder was present, sampling began 250 pm away from the shoulder (measured along the internal elastic lamina). A l l slides were blinded by a colleague for quantitative analysis. 47 Chapter 3: Alterations in Atherosclerotic Plaque Organization Table 3.3. Sample distribution from the abdominal aorta of W H H L rabbits. :>vj"yjlS3 GOD 3032-3 (control) 40 4 3032-4 (control) 38 12 3032-5 (control) 40 19 3032-9 (control) 70 23 3032-10 (control) 28 5 2819-1 (control) 42 0 2864-2 (control) 59 16 3004-2 (control) 40 9 3032-1 (PMio) 27 0 3032-2 (PMio) 34 0 3032-6 (PMio) 40 24 3032-7 (PMio) 29 8 3032-8 (PMio) 34 11 2819-2 (PM,o) 42 13 2864-1 (PMio) 52 18 3004-1 (PMio) 38 0 Total 653 PMio = 74 Control = 88 Blinded slides were analyzed using a N i k o n Labophot-2 Light Microscope (Nikon, Mississauga, ON) and a eye piece with a built in ruler. The length o f the endothelium was measured and the total number of macrophage-derived foam cell profiles that fell within a depth of 23 pm from the abluminal surface of the endothelium was counted. If less than 50% of a foam cell profile was within the 23 pm region it was not counted. These measurements were made on the 0.5 um sections cut from 88 different T E M blocks from 7 control rabbits and 74 different T E M blocks from 5 P M ) 0 exposed rabbits that met the inclusion criteria described above. 3.4 Results 3.4.1 The Basis of the 23 pm Depth of Interest To ensure in an unbiased fashion that the population of quantified macrophage-derived foam cells was subendothelial and therefore capable of interacting with the plaque endothelium we defined the limits of the subendothelial region o f interest by measuring the diameter of 75 48 Chapter 3: Alterations in Atherosclerotic Plaque Organization randomly selected macrophage-derived foam cell profiles in section. Their mean diameter in section was 17.4 ± 5.8 um (mean ± SD). Therefore, for this study we established the limit of the depth of interest at 23 u m (mean + 1 SD) below the endothelium representing the region in which foam cells were considered to be in close enough proximity to interact with the endothelium and its immediate E C M substrate. 3.4.2 Qualitative Observations In the process of evaluating the slides for inclusion in the study, several distinct morphological features were noted. The arterial wall in non-diseased areas appeared normal, with a thin intima, a robust media of S M C s and an adventitia composed o f loose connective tissue (Figure 3.1). With our T E M fixation protocols, the delicate endothelium of the intima was preserved intact and clearly visible even with light microscopy (Figure 3.1b). Figure 3.1. The three layers of a healthy arterial wall, a) The intima, the media and the adventitia. The media was thick and composed of alternating layers of smooth muscle cells and elastin. The adventitia was made of connective tissue. Scale bar = 50 pm. b) The intima consisted of a single layer of endothelial cells (*) on the internal elastic lamina (between arrowheads). Scale bar =10 pm. The atherosclerotic plaques from control rabbits were mostly confined to the intima, and separated from the media by an intact internal elastic lamina (Figure 3.2a and 3.2e). The control plaques had prominent sequestrations of lipid within the plaque cores deep below the endothelium (Figure 3.2a, 3.2c and 3.2e), with cholesterol crystals observed deep within the 49 Chapter 3: Alterations in Atherosclerotic Plaque Organization cores o f these control plaques in among areas o f l ipid and necrotic tissue debris (Figure 3.2c). In some cases a distinct layer of macrophage-derived foam cells was observed within the core regions (Figure 3.2e). A distinct E C M , S M C rich fibrous cap was observed between the endothelium and the l ipid core (Figure 3.2). Within the fibrous cap, S M C s and occasional macrophages were observed below the endothelium; both cell types had minimal l ipid accumulations (Figure 3.2b, 3.2d and 3.2f). These atherosclerotic plaques from control W H H L rabbits exhibited the characteristics of type V atherosclerotic plaques. L ike the atherosclerotic plaques from control rabbits, atherosclerotic plaques from the PMio exposed rabbits were largely confined to the intima, separated from the media by the intact internal elastic lamina (Figure 3.3a, 3.3b and 3.3e). However, in contrast to the plaques from control rabbits, the plaques from PMio exposed rabbits no longer had a distinct deep l ipid core. Rather, these plaques had l ipid deposits and sparse macrophage-derived foam cells distributed throughout the atherosclerotic plaque. The only consistently distinctive layer o f lipids and cells observed in the plaques from PMio exposed rabbits was an accumulation of macrophage-derived foam cells subtending the endothelium (Figure 3.3a, 3.3c and 3.3d). Occasionally, mechanical disruption o f the surface of these plaques was observed. These disruptions occurred in regions of accumulations of l ipid laden macrophage-derived foam cells immediately below the endothelium (Figure 3.3e and 3.3f). Smooth muscle cells were still observed with moderate amounts o f l ip id accumulation; however, they were now mostly separated from the endothelial cells by the layer of macrophage-derived foam cells (Figure 3.3). Wi th the high quality of glutaraldehyde fixation for T E M , macrophage-derived foam cells could readily be differentiated from smooth muscle cell derived foam cells (Figure 3.4). The S M C derived foam cells stained darker with T B O than the macrophage-derived foam cells (Figure 3.4). In addition, S M C s appeared more spindle shaped in long section, and smaller in cross section than the macrophage-derived foam cells (Figure 3.4). Furthermore, lipids were contained in the center of the S M C derived foam cells whereas the lipids in the macrophage-derived foam cell came to fill the entire cell volume except the nuclei (Figure 3.4). 50 Chapter 3: Alterations in Atherosclerotic Plaque Organization Figure 3.2. Atherosclerotic plaques from control W H H L rabbit abdominal aortas, a) A plaque with a necrotic core (*) just above the internal elastic lamina (arrowheads) of the media, a fibrous layer o f S M C s and E C M separate the necrotic core from the endothelium. Scale bar = 50 pm. b) Inset from panel a. Small l ipid deposits visible in S M C derived foam cells (arrow). Scale bar = 10 pm. c) Plaque with an extensive necrotic core (*) and crystalline cholesterol deposits (white arrows). Cap regions were populated by S M C s and macrophages with minimal lipid accumulation. Scale bar = 50 pm. d) Inset from panel c. showing a thick fibrous cap of S M C s (arrows) and E C M (arrowheads). Scale bar = 10 pm. e) Plaque with a distinct l ipid core containing macrophage-derived foam cells and an overlying distinct fibrous cap region. Scale bar = 50 pm. f) Inset from e. E C M (arrowheads) surrounding non lipid filled cells (arrows) in the fibrous cap of control plaque. Scale bar = 10 pm. 51 Chapter 3: Alterations in Atherosclerotic Plaque Organization Figure 3.3. Atherosclerotic plaque from PMio exposed W H H L rabbit abdominal aortas. a) Exposed plaque with macrophage-derived foam cells accumulated below the endothelium and an acellular core, b) Plaque with macrophage-derived foam cells sparsely dispersed throughout with some subendothelial accumulation (*). c) A plaque with a necrotic core (*). Macrophage-derived foam cells and S M C s throughout the plaque, and an accumulation of macrophage-derived foam cells between the endothelium and the fibrous core, d) Inset from panel c. Macrophage-derived foam cells subtending the endothelium and remnants of a S M C fibrous cap over a necrotic core (*). e) Disrupted endothelial surface and foam cells distributed throughout the depth of the plaque, f) Inset from panel e. Disrupted endothelium and macrophage-derived foam cells of the subendothelial region. A l l scale bars = 50 pm. 52 Chapter 3: Alterations in Atherosclerotic Plaque Organization Figure 3.4. Macrophage-derived foam cells and S M C derived foam cells in the upper cap region of an atherosclerotic plaque from a PMio exposed W H H L rabbit. The S M C derived foam cells are present in long and cross section (*) and stained darker with T B O than the macrophage-derived foam cells (MO) . Furthermore, lipids are confined to the center of the S M C derived foam cells whereas the lipids in the macrophage-derived foam cell fill the entire cell. Scale bar = 10 pm. 3.4.3 Quantitative Results Analysis of the number of macrophage-derived foam cells within 23 p m of the endothelium revealed a statistically significant increase in the size of the macrophage-derived foam cell population accumulating immediately subtending the endothelium in the cap regions of PMio exposed rabbits. PMio exposed rabbits had an average of 96 foam cells per mm of endothelium (95% confidence interval 80 - 110 foam cells) compared to only 41 foam cells per mm of endothelium (95% confidence interval 35 - 4 8 foam cells) in the control rabbits (p = 0.04) (Figure 3.5). 3.5 Discussion The observations described in this light microscopic investigation demonstrate a statistically significant accumulation of macrophage-derived foam cells immediately below (within 23 pm of) the plaque endothelium of W H H L rabbits in response to repeated exposure to PMio. 53 Chapter 3: Alterations in Atherosclerotic Plaque Organization Qualitative observations showed organizational changes to the atherosclerotic plaques of PMio exposed W H H L rabbits evidenced by reduced plaque stratification, an absence of a distinct necrotic core and increased macrophage-derived foam cells subtending the endothelium. Control PMIO Figure 3.5. Quantification of the subendothelial population of macrophage-derived foam cells. Nested A N O V A p = 0.04. Previous research by Suwa and colleagues 1 0 6 demonstrated larger overall plaque size, increased l ipid accumulation and increased inflammatory cell infiltration in atherosclerotic plaques from W H H L rabbits following PMio exposure. However, that study did not determine the exact, cellular or extracellular, location of accumulated l ipid, nor did it address the distribution o f the inflammatory cells throughout the plaque. A separate study by Sun and colleagues 1 0 7 extended the findings by Suwa and colleagues 1 0 6 with descriptions of an increased population of macrophages in the intimal and medial regions of atherosclerotic plaques from A p o E -/- mice fed high fat chow and exposed to low levels of PM2.5. Our observations extend the findings of both of these previous studies by providing a more detailed description of the localization o f macrophage-derived foam cells beneath the endothelium, reduced atherosclerotic plaque stratification, and an absence of a distinct necrotic core from rabbits exposed to PMio. 54 Cnapter 3: Alterations in Atherosclerotic Plaque Organization The accumulation of a concentrated population of macrophage-derived foam cells immediately below the endothelium o f the plaque could have many important consequences and raises numerous questions, such as what are the consequences to endothelial cell adhesion and function in areas of macrophage-derived foam cell invasion? What was the origin of the subendothelial macrophage-derived foam cells, the systemic circulation or other regions of the atherosclerotic lesion? What are the implications of macrophage-derived foam cell migration within atherosclerotic plaques on plaque stability? The cytokine production capabilities of macrophage-derived foam cells are known to be very important in monocyte and neutrophil recruitment into the plaque; E C M degradation; and altered functions of the i "\n oo "X oo A. endothelium, ' ' all features of advancing and destabilizing atherosclerotic plaques. Furthermore, the importance of endothelial dysfunction in atherosclerotic plaque formation is well established. " The maintenance of endothelial cell metabolism and function are ooa OOQ partially dependent on cell to cell contact and cell to E C M adhesion, leading to the suggestion that i f the macrophage-derived foam cells subtending the endothelium are degrading the E C M , the adhesion and function of the endothelium may also be compromised. In this context, the association o f areas of mechanical damage during processing with areas of macrophage-derived foam cells subtending the endothelium is not surprising. The mechanical damage was an artefact, but we would suggest that the presence of the macrophage-derived foam cells made the tissue more vulnerable to the mechanics o f the processing protocol. Degradation or reorganization of the E C M subtending the endothelium would compromise the stability of the endothelial attachments to basal E C M and neighbouring cells alike. Our novel observation of an increased population of macrophage-derived foam cells below the endothelium constitutes a significant contribution to the understanding o f how chronic exposure to PMio alters atherosclerotic plaque organization. The primary focus of the work described in subsequent chapters was to use the high resolution o f electron microscopy to identify and characterize changes in the organization of the atherosclerotic plaque, in order to understand the potential consequences of the accumulation of macrophage-derived foam cells in the subendothelial region. 55 CHAPTER 4 Endothelial Cell Contacts 4.1 Introduction The endothelium is a heterogeneous single layer of squamous cells that line arteries, capillaries and veins, weighs approximately 1 kg and covers an area in excess of several square meters. 2 3 0 Observations made using electron microscopy in the 1950s laid the foundation for the current perspective that the endothelium is a dynamic organ that has immunologic, secretory, synthetic and metabolic functions. 2 3 1 A s the barrier between circulating blood and surrounding tissues, tightly controlled endothelial permeability regulates the movement of nutrients, wastes and cells across the blood vessel w a l l . 2 3 0 The expression o f appropriate adhesion molecules, such as V C A M - 1 and I C A M - 1 on the endothelial surface is essential for selective leukocyte transmigration across the 1-3-5 O O ' J , endothelium. ' In addition, the endothelium contributes to the regulation of blood pressure and blood flow by producing and releasing vasoconstrictors, including endothelin (ET) and platelet-activating factor (PAF) , as well as vasodilators such as prostacyclin and 'yj-z 9 1 A N O . ' Furthermore, the antithrombotic surface of the endothelium inhibits platelet adhesion and clotting thereby facilitating continued blood f l o w . 2 3 0 A l l o f these functions o f the endothelium are critical to our health and survival; any alteration in the ability of the endothelium to perform any of these functions w i l l compromise the health of the whole organism. In healthy arteries the endothelium is situated on a thin E C M layer o f discontinuous basal 9 ^ 9 O'X'X lamina that provides vital support for endothelial cell attachment, morphogenesis, ' p ro l i fera t ion , 2 2 8 ' 2 3 4 migra t ion , 2 3 4 - 2 3 6 s u r v i v a l , 2 3 3 ' 2 3 7 ' 2 3 8 and even blood vessel stabilization. 2 2 9 The complex mechanisms through which the basal lamina facilitates endothelial cell functions include both external structural support as well as the internal regulation o f 56 Chapter 4: Endothelial Cel l Contacts intracellular signalling pathways some o f which include apoptosis, cytoskeletal reorganization, endothelial cell proliferation, 2 3 9 " 2 4 1 and endothelial cell shape. 2 4 2" 2 4 4 Although angiogenic cytokines such as vascular endothelial growth factor are known to be potent stimulators of endothelial cell proliferation, migration, and survival, a growing body of evidence suggests that the E C M substrate subtending the endothelium has an equal or greater 9^ 9 947 effect on the fundamental functions and integrity o f the endothelium. ' The basal lamina observed subtending the endothelium in healthy vasculature has been replaced in atherosclerotic plaques by a thicker layer of E C M elements including collagens, elastins and glycoproteins (such as laminin) . 2 4 8 Though the ultrastructural organization of E C M in atherosclerotic plaques differs, it is thought to retain similar functions to the E C M in 948 the healthy vasculature. The amount of E C M deposition in an atherosclerotic plaque must be carefully controlled. Excess deposition causes narrowing of the blood vessel lumen and a restriction of blood flow; however, inadequate deposition leads to an increased risk of atherosclerotic plaque rupture. 2 4 9 Thus a very fine balance is essential to prevent atherosclerotic plaque rupture without excessively narrowing the blood vessel lumen and drastically altering vascular hemodynamics. In Chapter 3, the observed presence o f macrophage-derived foam cells immediately below the endothelium dramatically altered the organization of the cap. Macrophage-derived foam cells are known to secrete M M P s , enzymes involved in E C M breakdown and plaque * * 224 250 251 reorganization. ' ' The presence o f the macrophage-derived foam cells subtending the endothelium may have compromised the organization of the subendothelial E C M . Decreased endothelial anchoring to an underlying E C M substrate increases the risk o f atherosclerotic plaque erosion and rupture. 2 4 9 The primary goal of this study was to determine whether the basal lamina is either digested or displaced from below the endothelium. In this study, qualitative and quantitative observations were made using electron microscopy on endothelial contact with E C M in atherosclerotic plaques from control and PMio exposed W H H L rabbits. 3-dimensional reconstruction was used to confirm the observed changes to the architecture of the reticulum 57 Chapter 4: Endothelial Ce l l Contacts of dense E C M that subtends the plaque endothelial cells in the PMio exposed rabbits. The observations made in this study require the resolution of the transmission electron microscope. 4.2 Aims 1. To define the organization of the E C M upon which the plaque endothelium sits in the control W H H L rabbits and compare that with the organization of the E C M in PMio exposed W H H L rabbits where macrophage-derived foam cells have accumulated in the subendothelial region. 2. To quantitatively assess differences in the area o f endothelial cell / dense extracellular matrix contacts in atherosclerotic plaque caps o f PMio exposed and control W H H L rabbits. 4.3 Methodology Common exposure and tissue processing protocols were outlined in Chapter 2. The following protocols were carried out on the epon embedded blocks destined for T E M analysis. 4.3.1 Qualitative Observations From the same blocks of tissue used in the foam cell quantification, 70 nm thin sections were cut on the Leica E M U C 6 microtome and picked up on formvar coated slot grids. Thin sections were stained for 4 minutes each with uranyl acetate and lead citrate. A l l sections were imaged using an F E I Tecnai 12 Transmission Electron Microscope. T E M observations required in excess of 100 hours of examination time during which more than 2,100 images were captured. While most observations and images came from the cap regions o f the atherosclerotic plaques, some time was also spent investigating the core and shoulder regions of the plaques. 4.3.2 Morphometric Analysis Morphometric T E M analysis was done on all samples used for the L M analysis. Images were captured in a systematic random fashion at 9,700x magnification. Briefly, the first image was 58 Chapter 4: Endothelial Ce l l Contacts acquired two fields o f view from the left edge of the tissue section and, i f possible, five images were acquired per block (63 control and 74 PMio blocks) with two fields of view separating each image. Images were not acquired in areas of mechanical damage, absent endothelium, or staining artefacts and we proceeded to the next location two fields beyond to continue sampling. For analysis, a cycloid grid mask was placed over the images parallel to the endothelium (Figure 4.1) and the line intercept method was used to quantify the frequency of endothelial contacts (within 15 nm) with the dense extracellular matrix (Figure 4.2), fragmented Figure 4.1. Sample transmission electron micrograph from a control plaque with a superimposed sigmoid grid mask. Two macrophage-derived foam cells are separated from the endothelium by a reticulum of dense E C M . In each location where the red sigmoid line crossed the abluminal surface (arrows) it was noted what the endothelium was in contact with at a distance of 15 nm or less. Scale bar = 2 pm. 5 9 Chapter 4: Endothelial Ce l l Contacts extracellular matrix (Figure 4.3), macrophage (Figure 4.4), macrophage-derived foam cell (Figure 4.5), S M C (Figure 4.6), smooth muscle cell derived foam cell (Figure 4.7), unknown structures (Figure 4.8) and space (Figure 4.9). 297 images, with 2,791 intersects, were analyzed for the control atherosclerotic plaques and 357 images, with 3,297 intersects, were analyzed for the PMio exposed atherosclerotic plaques. The total number of images differed due to inadequate tissue fixation o f one control rabbit (3032-9) that resulted in that rabbit being removed from the morphometric analysis. However, the mean number of intersects per micrograph for the controls was 9.40 and for the PMio exposed the mean was 9.24 (Figure 4.10). The similarity of these two means demonstrates an absence of a sampling bias, or an altered size, shape or surface morphology in endothelial structure between the two groups of images analyzed. Figure 4.2. Dense extracellular matrix subtending endothelial cells (EC). The dense E C M organized as a reticulum in W H H L rabbit atherosclerotic plaques. Scale bar = 2 pm. 60 Chapter 4: Endothelial Cel l Contacts Lumen Figure 4.3. Fragmented extracellular matrix subtending endothelial cells (EC). The upper left area under the endothelium has small focal areas of the electron dense E C M that do not appear to form a reticulum. Scale bar = 2 pm. Figure 4.4. Macrophage in direct contact with the abluminal surface of an endothelial cell (EC) (arrows). Scale bar = 2 pm. 61 Chapter 4: Endothelial Cel l Contacts E C % ' f t Figure 4.5. Macrophage-derived foam cell in direct contact with the abluminal surface of an endothelial cell (EC). Scale bar = 2 pm. Figure 4.6. A smooth muscle cell in direct contact with the abluminal surface of an endothelial cell (EC) (arrows). Scale bar = 2 pm. 62 Chapter 4: Endothelial Ce l l Contacts Lumen S M C Figure 4.7. Smooth muscle cell derived foam cell in direct contact with the abluminal surface of endothelial cells (EC) (arrows). Scale bar = 2 um. Lumen Figure 4.8. Unknown cell structures in direct contact with the abluminal surface of endothelial cells (EC). There appears to be a membrane surrounding the cell structures, and the internal environment is distinct from the outside environment although there are no discemable organelles inside the unknown structures to suggest that they are cells. Scale bar = 2 um. 63 Chapter 4: Endothelial Cel l Contacts Lumen Figure 4.9. Space subtending endothelial cells (EC). The macrophage-derived foam cells ( M O F C ) may separate the endothelium from underlying structures resulting in space below the endothelium. Scale bar = 2 pm. 4.3.3 3-Dimensional Reconstruction Eight serial reconstructions (n = 4 control and n = 4 PMio) of the dense E C M subtending the endothelium were done. Each reconstruction consisted of approximately 20 consecutive 100 nm thick sections of an atherosclerotic plaque. Images were captured from the sections at 19,500x and 37,000x magnifications. Images were then manually aligned and traced in Photoshop prior to rendering using a computer reconstruction program called T3D. Reconstructions done at 37,000x magnification gave adequate resolution to elucidate the organization of the E C M subtending the endothelium and became the focus of the reconstruction work. 4.4 Results 4.4.1 Qualitative Observations The intima of non-diseased regions of the abdominal aorta had an intact layer of endothelial cells resting on a thin basal lamina above the internal elastic lamina (Figure 4.11). Adherens 64 Chapter 4: Endothelial Ce l l Contacts 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Number of Intersects per Micrograph Figure 4.10. The number of intersects per micrograph analyzed in the morphometric analysis, a) A histogram o f the number of line intersects per micrograph, b) The mean number of intersects per micrograph for control was 9.40 and for the PMio exposed was 9.24. type contacts anchored the endothelium to underlying basal lamina or the internal elastic lamina (Figure 4.1 l b and 4.1 lc) . Sporadic intimal S M C s were observed (Figure 4.1 Id). In the fibrous cap regions o f the control rabbit plaques, S M C s and a unique reticulum o f dense E C M were observed (Figures 4.12). Although not abundant, occasional macrophage-derived foam cells were observed. However, in spite of their presence, in most cases these cells were clearly separated from the abluminal endothelial surface by the reticulum o f dense E C M in control animals (Figure 4.1) or there were only small regions o f contact between the endothelium and macrophage-derived foam cells (Figure 4.13c and 4.13d). In the PMio exposed plaques, there were macrophage-derived foam cells in the cap region and a concomitant absence (Figure 4.14) or fragmentation (Figure 4.15) of the reticulum of dense E C M elements immediately beneath the endothelial surface. Foam cells were observed at varying distances below the endothelium and in many areas direct contact between the 65 Chapter 4: Endothelial Ce l l Contacts endothelium and the macrophage-derived foam cells was observed. Though contact between macrophage-derived foam cells and endothelial cells was observed in both control and PMio exposed rabbits, the extent of contact in the plaques from PMio exposed rabbits was much greater than the extent o f contact in control rabbits (Figure 4.13d versus Figure 4.15d). Figure 4.11. Intima of non-diseased abdominal aorta a) The intima includes the endothelium (EC), internal elastic lamina (IEL) and a narrow space between. Wrinkles in the I E L (arrowhead) are artefacts. Scale bar = 2 pm. b) Inset from panel a. Adherens contacts between the E C and the I E L (black arrows). Scale bar = 0.2 pm. c) Basal lamina between the E C and the IEL (white arrows). Scale bar = 0.2 pm. d) A n intimal S M C between the endothelium and the IEL. Scale bar = 2 pm. 6 6 Chapter 4: Endothelial Cel l Contacts Figure 4.12. Fibrous cap of a stable plaque in control W H H L rabbit, a) The endothelium of control W H H L rabbits rests upon a fibrous cap consisting principally o f a unique reticulum of dense E C M populated by smooth muscle cells. Scale bar = 5 um. b) Detail o f unique reticulum of electron dense E C M in panel a. Scale bar = 1 pm. During the investigation o f the core, shoulder, and cap regions o f atherosclerotic plaques from both control and PMio exposed rabbits, two primary cell types were observed: the smooth muscle related cells and the macrophage related cells, though the occasional lymphocyte was also observed (Figure 4.16). There were two primary macrophage related cell types unevenly distributed throughout the atherosclerotic plaques from both the control and the PMio exposed rabbits. The first cell type was the tissue macrophage. These cells accounted for a very small number of the total cell population. They were between 4 and 8 p m in cross sectional size (Figure 4.14) and had a central nucleus that occupied the majority of the cell with little to no l ipid deposition in the cytoplasm. This population o f cells was most frequently observed at or near the luminal surface of the atherosclerotic plaques from both the control and PMio exposed animals and was rarely seen in deeper regions of atherosclerotic plaques. Occasional lymphocytes were observed in direct contact with the endothelial cells (Figure 4.16). 67 Chapter 4: Endothelial Ce l l Contacts Figure 4.13. Architecture of control atherosclerotic plaques, a) Control plaque wall containing reticulum o f dense E C M subtending the endothelium (EC). L ip id droplets are visible in the endothelial cells and the macrophage-derived foam cell (FC). Scale bar = 2 pm. b) Reticulum of dense E C M extends from the endothelium down to the macrophage-derived foam cell. Scale bar = 1 pm. c) Contact does occur between the foam cell and the endothelium, though the overall contact area is small. Scale bar = 2 pm. d) Inset of contact between the E C and the macrophage-derived foam cell (between arrows). Scale bar = 0.5 pm. 68 Chapter 4: Endothelial Cel l Contacts Figure 4.14. Architecture of fibrous atherosclerotic plaques from a PMio exposed rabbit. a) When macrophage-derived foam cells and macrophages were present in the cap region, the reticulum of electron dense E C M was fragmented. Scale bar = 2 pm. b) A macrophage-derived foam cell in close proximity to the endothelium loaded with large l ipid deposits including a cholesterol crystal (arrow). Scale bar = 2 pm. The macrophage-derived foam cells were the second population of macrophage related cells and they were also the most commonly observed macrophage phenotype. These cells had accumulated large amounts of lipid becoming 2 to 3 times larger than the observed macrophages (Figure 4.17). They were approximately 17 p m in diameter (Chapter 3). Although this population o f cells was observed throughout the plaque, their distribution differed dramatically between control atherosclerotic plaques and the PMio exposed plaques. While the control animals had a larger population of the macrophage-derived foam cells deep in the cores of the plaques, the P M ] 0 exposed plaques had these cells throughout the plaque with a large population accumulated in the subendothelial cell region. Macrophage-derived foam cells were infrequently observed adherent to the luminal surface o f the atherosclerotic plaques (Figure 4.18) or transmigrating through the endothelium (see Chapter 6 for further details). 69 Chapter 4: Endothe l ia l C e l l Contacts Figure 4.15. Architecture of an atherosclerotic plaque from a PMio exposed rabbit, a) L i p i d engorged macrophage-der ived foam cel ls immedia te ly subtending the endothe l ium. Scale bar - 5 p m . b) Inset f rom the upper box in a. The macrophage-der ived f oam ce l l is c lear ly v is ib le subtending the entire v i s ib le length o f an endothel ia l ce l l . There is no E C M between the endothel ia l ce l l and the foam ce l l . Sca le bar - 1 p m . c) A macrophage-der ived foam ce l l subtending a st i l l intact junc t ion between two endothel ia l cel ls . Sca le bar = 1 p m . d) Deta i led image o f direct contact between the macrophage-der ived foam ce l l and an endothel ia l ce l l . The cy top lasmic extensions (*) o f the macrophage-der ived foam ce l l fo lded together d i rect ly be low the endothel ia l ce l l and in direct contact w i th the endothel ia l ce l l . Th is image is f rom the bot tom box in panel a. Scale bar = 1 p m . 70 Chapter 4: Endothelial Ce l l Contacts Figure 4.16. Lymphocyte in contact with the endothelium of a control atherosclerotic plaque a) Lymphocyte with multiple contacts with the endothelium. Intact reticulum of dense E C M surrounding the lymphocyte. Scale bar = 2 um. b) The arrows point to an endothelial extension extending down the right side of the cell. Scale bar = 1 pm. c) Another extension of another endothelial cell extends into the plaque and contacts the lymphocyte directly and then extends further up the side of the lymphocyte (as outlined by the arrows). Scale bar = 1 pm. Chapter 4: Endothelial Ce l l Contacts Figure 4.17. Macrophage-derived foam cells in an atherosclerotic plaque, a) A group of cells exhibiting the defining characteristic o f a macrophage-derived foam cell: heavy lipid load occupying the majority of the cytoplasm leaving just enough room for the nucleus. Scale bar = 2 pm. b) Inset from panel a. The macrophage-derived foam cell has an absence of a basal lamina surrounding the cell and an accumulation o f lipid particles occupying the cell cytoplasm right up to the plasma membrane, both features not associated with smooth muscle cell derived foam cells. Scale bar = 1 pm. Three distinct populations of smooth muscle cells were observed within the atherosclerotic plaques. The first cell type was a typical actin rich smooth muscle cell. These cells were very electron dense, appearing almost black when imaged using T E M (Figure 4.19a). A t a higher magnification actin could be observed in the very electron dense cells (Figure 4.19b). These cells were typically surrounded by an electron dense basal lamina and irregular E C M deposits (Figure 4.19a and 4.19b). A small proportion of these actin rich cells had accumulated large deposits o f lipid to become lipid engorged S M C derived foam cells (Figure 4.20). The smooth muscle cell derived foam cells were distinguishable from the macrophage-derived foam cells by the presence of their thin basal lamina (Figure 4.21) and the confinement of accumulated l ipid to the center of the cell by the extensive peripheral cytoskeletal apparatus (Figures 4.20, 4.21 and 4.22). There was also evidence of S M C derived foam cell attachment to elastin in the atherosclerotic plaques (Figure 4.22c). A third population of S M C s was characterized by large amounts of rough endoplasmic reticulum (RER) throughout the entire cell (Figure 4.23) and surrounded by an extensive 72 Chapter 4: Endothelial Cel l Contacts reticulum of dense E C M . Despite their distinctive, conspicuous appearance, these cells rich in R E R were not frequently observed. Figure 4.18. A macrophage-derived foam cell at the shoulder of a control atherosclerotic plaque, a) Adherent to the surface o f several macrophages is a l ipid engorged macrophage-derived foam cell in a gap between two endothelial cells (EC). Scale bar = 5 um. b) Inset shows the area of macrophage-derived foam cell attachment on the surface o f a series of tissue macrophages (*). 73 Chapter 4: Endothelial Cell Contacts Figure 4.19. Actin rich smooth muscle cells in control plaques, a) A low magnification image of actin rich SMC subtending the endothelium. Scale bar = 2 pm. b) Smooth muscle cell rich in cytoskeletal apparatus surrounded by a reticulum of electron dense E C M . Scale bar = 1 pm. Figure 4.20. Smooth muscle cell derived foam cells, a) A characteristic smooth muscle cell derived foam cell with lipid sequestered in the core of the cell by the cytoskeletal apparatus separating the lipid from the plasma membrane. Scale bar = 2 pm. b) The smooth muscle cell derived foam cell is surrounded by a dense E C M , elastin and extracellular lipid deposits (*). Scale bar = 1 pm. 74 Chapter 4: Endothe l ia l C e l l Contacts Figure 4.21. Smooth muscle cell derived foam cell, a) A S M C der ived foam c e l l surrounded b y extensive E C M . Scale bar = 2 p m . b) L i p i d deposits and nucleus are v i s ib l e i n the c e l l and sma l l extracellular l i p i d droplets are v i s ib le i n the surrounding E C M . Scale bar = 1 p m . c) A thin layer o f basal l amina ( B L ) extends around the perimeter o f the ce l l . Scale bar = 1 p m . 75 Chapter 4: Endothelial Cel l Contacts Figure 4.22. Smooth muscle cell derived foam cell, a) S M C derived foam cell surrounded by irregular E C M and extracellular lipid in a plaque of a PMio exposed rabbit. Scale bar = 2 pm. b) The cell in a. is showing cytoplasmic extensions around E C M elements, possibly elastic fibres. Scale bar = 1 pm. c) A higher magnification of the left side of the cell with two cross sections of elastin bundles (arrowheads). A reticulum of electron dense E C M with extensive lipid droplets surrounding the S M C derived foam cell. Scale bar = 1 pm. d) Extracellular lipid (arrows) that are about 200 nm in size interspersed in the E C M of the cap of the atherosclerotic plaque and in close association to elastin fibres (arrowhead). Scale bar = 0.5 pm. 76 Chapter 4: Endothelial Ce l l Contacts Figure 4.23. Synthetic smooth muscle cells, a) A smooth muscle cell filled with rough endoplasmic reticulum (RER) and surrounded by a reticulum of dense E C M . Scale bar = 2 pm. b) Detail of R E R in panel a. Scale bar = 1 pm. c) A cap region smooth muscle cell filled with similar R E R and surrounded by dense E C M . The endothelium is visible on the left side of the image and marked by an asterisk (*). Scale bar = 2 pm. d) The extensive R E R o f the S M C . Scale bar = 1 pm. In plaques from both PMio exposed rabbits and control rabbits, S M C s located deeper within the atherosclerotic plaque core typically had less l ipid accumulation than cells near the abluminal endothelial surface. S M C s within the core of the plaque tended to be oriented so that their long axis was perpendicular to the endothelium, (Figure 4.24) whereas SMCs near 77 Chapter 4: Endothelial Ce l l Contacts the luminal surface, often within the fibrous cap, were oriented so that their long axis was parallel to the endothelium (Figure 4.19). There were also qualitative observations o f differential distribution of S M C s between the PMio exposed rabbits and control rabbits. The upper cap regions o f control atherosclerotic plaques were dominated by S M C s and E C M . B y comparison the plaques from PMio exposed rabbits had the S M C population scattered throughout the plaque without any particular distinctive location of S M C s . Some PMio exposed plaques had a S M C band located below the subendothelial macrophage-derived foam cells; however, this was not a consistent observation. N o ruptured atherosclerotic plaques were observed in either the control or PMio exposed rabbits. Free l ipid was observed in both control and PMio exposed rabbit plaques. Abundant intercellular deposits were also seen in macrophage-derived foam cells, S M C derived foam cells and endothelial cells. O f the three cell types, more intracellular l ipid deposits were qualitatively observed in the macrophage-derived foam cells whereas endothelial cells routinely showed the lowest levels of l ip id deposition. L ip id droplets typically appeared lightly electron dense occasionally containing electron dense material (Figures 4.17, 4.21 and 4.22). On the odd occasion, the l ipid deposits were very electron dense appearing black in micrographs and exhibiting a lamellar organization of the electron dense material (Figures 4.13). In macrophage-derived foam cells with extensive l ipid deposition, cholesterol crystals were periodically observed. In the control atherosclerotic plaques, the cells containing cholesterol crystals were observed deep within the cores of atherosclerotic plaques. In contrast, the PMio exposed plaques had a primary population o f macrophage-derived foam cells with cholesterol crystals located deep in the plaque, and a second population located in the upper regions of the atherosclerotic plaque near the endothelium (Figures 3.3 and 4.14b). Small droplets of extracellular l ipid were observed among E C M elements throughout plaques (Figure 4.20d). In the core regions of some plaques, much larger extracellular l ipid droplets and cholesterol crystals were observed (Figure 4.25). 78 Chapter 4: E n d o t h e l i a l C e l l Contacts Figure 4.24. Smooth muscle cell orientation in an area of the media and central core of an atherosclerotic plaque. T h e S M C s deep i n the m e d i a are oriented para l le l to the endothel ium. B y contrast, the S M C s i m m e d i a t e l y b e l o w the internal elastic l a m i n a ( I E L ) and i n the core reg ion are oriented so their l o n g axis is perpendicular to the endothel ium (*). Scale bar = 2 p m . Chapter 4: Endothelial Ce l l Contacts Figure 4.25. Extracellular lipid deposition in the core of an atherosclerotic plaque, a) Low magnification of extracellular lipid droplets and cholesterol crystals. Scale bar = 5 pm. b) Higher magnification of the cluster of l ipid droplets o f all sizes. Scale bar = 2 pm. In one atherosclerotic plaque from a control rabbit unknown structures were observed in several macrophage-derived foam cells deep within the core of the plaque (Figure 4.26). These structures had bands o f electron dense material alternating with bands of non-electron dense material resulting in a lamellar myelin like appearance. A t 96,000x magnification the precise organization o f the structures is apparent. The structures appear to be composed of lipid, though the exact nature of the structures remains unknown. We have called them zebra bodies to represent their zebra like structure. 4.4.2 Morphometric Analysis Structural features usually associated with the stable endothelial attachment were found to be compromised in the PMio exposed rabbits (Figure 4.27). For example, in the PMio exposed rabbits the frequency of the endothelial to dense E C M contacts was only 21.3% of the 3297 endothelial intersections quantified (p < 0.0001). In marked contrast, in control rabbits, 65.7% of the 2791 endothelial intersections observed by line intercept were contact areas between the endothelium and the reticulum of dense E C M . Since contacts between endothelial cells and S M C were rare in both control and PMio atherosclerotic plaques, no 80 Chapter 4: Endothelial Cel l Contacts significant change in the frequency o f endothelial intersects with S M C s was found (Figure 4.27). Figure 4.26. Zebra bodies within macrophage-derived foam cells located in the core of an atherosclerotic plaque, a) A macrophage-derived foam cell containing two odd structures exhibiting a lamellar myelin like appearance. Scale bar = 5 pm. b) A more detailed image o f one o f the structures. Adjacent l ipid droplets are clearly visible, and different from the unknown structure. Scale bar = 1 pm. c) A more detailed image of the second unknown structure. The structure appears to be compartmentalized with a possible site o f lysosomal fusion (*). Scale bar = 1 pm. d) A 97,000x magnification of the structure in panel c. Two levels o f organized banding are visible in the cell. The first is the light / dark banding visible. Each band appears to be about 150 nm thick. M u c h smaller banding is also visible in each light and dark section. Scale bar = 100 nm. 81 Chapter 4: Endothelial Ce l l Contacts When considering features common to a destabilized plaque, it was observed that in PMio exposed rabbits, 21.6% of 3297 endothelial cell contacts were with fragmented E C M , whereas only 8.4% o f 2791 endothelial contacts in control rabbits were in contact with fragmented E C M (p < 0.0001) (Figure 4.27). The number of endothelial cell contacts with macrophage-derived foam cells was found to be 13.4% in the control rabbits and increased significantly to 38.1% in PMio exposed rabbits (p = 0.0039). The final quantified contact found to be significantly different between the control and PMio exposed rabbits, was the number of endothelial cell contacts with apparently empty space, observed to be only 7.6% in control and increased to 14.2% in PMio exposed plaques (p < 0.0001). The increased space subtending the endothelium could be a logical consequence of large macrophage-derived foam cells insinuating themselves between endothelial cells and the reticulum of dense E C M elements o f the cap region. Smooth Dense Smooth Fragmented Macrophage Macrophage Space Unknown Muscle Cell Extracellular Muscle Extracellular Foam Cell Structure Matrix Foam Cell Matrix Figure 4.27. Relative percentage of endothelial cell abluminal membrane contacts. Percentage o f endothelial cell contacts (< 15 nm) with subtending cells or substrate elements for control rabbits (n = 7) and PMio exposed rabbits (n = 5). Poisson regression analysis confirms PMio exposed rabbits have significantly less contact with dense E C M than control rabbits (* p-value < 0.0001) and significantly more contact with macrophage-derived foam cells than control rabbits (** p-value = 0.0039). 82 Chapter 4: Endothelial Ce l l Contacts It appears that as contact with the reticulum of dense E C M decreased significantly, contact with macrophage-derived foam cells, fragmented E C M and space all increased in the atherosclerotic plaques of PMio exposed W H H L rabbits. This is in agreement with our earlier observation of macrophage-derived foam cells accumulating under the endothelium of atherosclerotic plaques (Chapter 3). A l l other measured parameters, specifically contact with macrophages, smooth muscle cell derived foam cells and unknown structures, had comparable contact frequencies between the control and the PMio exposed tissues; however, this may be due to their small numbers, even in the control plaques. 4.4.3 3-Dimensional Reconstruction 3-dimensional reconstructions were done to directly demonstrate the extent of contact between the reticulum of dense E C M and the abluminal endothelial plasma membrane to compliment the estimates made using the line intercept technique. Observations from these 3-dimensional reconstructions confirmed that rabbits exposed to PMio showed substantially less contact between the reticulum of dense E C M and the endothelium when compared to control rabbits. Contact was reduced not from a separation o f the endothelium from the reticulum of dense E C M , but rather an absence o f the reticulum in the PMio exposed rabbits apparently due to its degradation and fragmentation. In the 3-dimensional reconstructions, the E C M subtending the endothelium in the plaques from control rabbits was abundant (Figure 4.28) and formed a reticulum o f basal lamina like material as opposed to a solid sheet. Each individual section had gaps in the E C M reticulum; (Figure 4.28b) however, when the reconstruction was rendered into a 3-dimensional model, the gaps were actually small spaces between the elements of the E C M reticulum that extended only through several sections and not the entire 20 sections o f the reconstruction (Figures 4.28c and 4.29a). Further to this, the E C M subtended the endothelium so tightly that when the endothelial cell was subtracted from the reconstruction, the shape and surface features of the endothelial cell were clearly reflected in the surface of the reticulum o f dense E C M (Figure 4.29). 83 Chapter 4: Endothelial Ce l l Contacts Figure 4.28. Derivation of a control serial 3-dimensional reconstruction of the abluminal extracellular matrix, a) A sample image captured at 37,000x magnification from the control reconstruction series, b) The tracing done from the image in a. c) The 3-dimensional reconstruction o f 20 such sections, green represents the E C M and orange represents the endothelial cell. B y contrast the reconstructions of the electron dense E C M subtending the endothelium in the atherosclerotic plaques from PMio exposed rabbits had much less E C M than the reconstructions done from control rabbit tissues (Figure 4.30). In the PMio exposed tissues, there was no longer an interconnected reticulum of dense E C M , rather small focal areas of electron dense material were observed in amongst fragmented E C M type material (Figure 4.30a) that had a similar appearance to the fragmented E C M quantified in contact with the endothelium in the morphometric analysis (Figure 4.3). Only the electron dense material was reconstructed in both control and PMio tissues leaving large areas o f fragmented E C M in the PMio atherosclerotic plaque tissue to appear as large gaps in the reconstructed E C M . The alignment of these gaps throughout the 19 100 nm sections amounts to reconstructed gaps of at least 1900 nm or 1.9 pm. When the endothelial cell was subtracted from the section, as done in the control reconstruction, the E C M subtending the endothelium in the PMio reconstruction no longer reflected the shape, size or surface morphology of the endothelial 84 Chapter 4: Endothelial Cel l Contacts cell with the exception of very large surface features (Figure 4.31). This is in agreement with our estimated decrease in endothelial contacts with the reticulum of dense E C M in the morphometric analysis. Figure 4.29. Topographical match between the endothelial basal plasma membrane and the reticulum of electron dense extracellular matrix in control atherosclerotic plaques. a) Reconstruction of 20 sections, green is the E C M and orange is the endothelial cell, b) Rotated perspective of reconstruction, endothelial cell is sitting on top o f the E C M . A ridge in the endothelial cell is marked by the white arrow, c) The endothelial cell has been subtracted exposing the underlying E C M surface outlined by the block arrows, d) Abluminal surface of the endothelial cell. The white asterisks are areas where topography illustrates extensive areas o f close contact (c & d). The white arrow is the same ridge of the endothelial cell that was noted in b. 85 Chapter 4: Endothelial Cel l Contacts Figure 4.30. Derivation of a PMio serial 3-dimensional reconstruction of the abluminal extracellular matrix, a) A sample image from reconstructed series showing obvious fragmentation of reticulum of electron dense E C M and decreased areas o f contact between the reticulum and the endothelial cell (EC), b) The tracing done from the image in a), c) The 3-dimensional reconstruction of 19 sections, green represents the E C M and orange represents the endothelial cell. When the PMio exposed and control reconstructions are viewed side by side, the much greater abundance of electron dense E C M elements in the control reconstruction is clear (Figure 4.32). S E M observations o f abdominal aortas revealed regions of eroded endothelium and platelets adherent to a reticulum of E C M elements also seen in the reconstructions. The S E M results w i l l be presented and discussed in Chapter 6. Refer to Figure 6.17d for a S E M micrograph of the dense E C M . 86 Chapter 4: Endothelial Ce l l Contacts Figure 4.31. PMio 3-dimensional reconstruction of the abluminal extracellular matrix / endothelial contact surface, a) Abluminal E C M surface that the endothelial cell was sitting on (black arrowheads), b) The abluminal surface of the endothelial cell. The white asterisks are located in areas that illustrate some degree of matching topography and areas of contact (a & b). Figure 4.32. Comparison of the control and PMio 3-dimensional reconstructions of the abluminal dense extracellular matrix, a) The control reconstruction o f 20 serial images, b) The PMio reconstruction o f 19 serial images. E C M fragmentation significantly reduces the area of endothelial cell / E C M contact and eliminates the topographical match between the basal plasma membrane of the endothelial cells with the fragmented reticulum of dense E C M . 87 Chapter 4: Endothelial Ce l l Contacts 4.5 Discussion In Chapter 3, we demonstrated that macrophage-derived foam cells accumulate beneath the endothelium of the central regions of atherosclerotic plaques in the aortas of W H H L rabbits exposed to PMio . T E M data presented in this chapter have shown that the insinuation of macrophage-derived foam cells immediately below the endothelium of atherosclerotic caps of PMio exposed rabbits was associated with a significant decrease in the support of the endothelium through the fragmentation and dissolution of the reticulum of dense E C M beneath it. This degradation of the supporting E C M was documented in both the 2- and 3-dimensional analysis, qualitatively and quantitatively undertaken in this chapter. To our knowledge these findings constitute the first demonstration of a potential mechanism whereby atherosclerotic plaques may become more unstable following PMio exposure. Changes o f this type could clearly lead to an increased risk of endothelial erosion or desquamation followed by atherosclerotic plaque rupture leading to thrombus formation. The importance of basal lamina in fundamental endothelial cell functions and survival has 9S9 been well documented in the literature. Characteristically the basal lamina is composed of type I V collagen and laminin arranged in a thin layer that surrounds cells of all types, including the endothelium. The ultrastructure of the reticulum of dense E C M observed subtending the endothelium in the atherosclerotic plaques of control W H H L rabbits has not been previously described. In the literature the basal lamina of rabbit atherosclerotic plaques has only been described as thicker than the basal lamina of a non-diseased blood vessel using 948 9^^ 9S4 occ light microscopic imaging. ' ' A 1989 paper by Nakamura and colleagues studied the effects of aging on atherosclerotic plaque development. Though they do not describe it, nor speculate on its composition or origin, a reticulum of dense E C M is visible in their electron micrographs in the atherosclerotic plaques from 18 month and 24 month old rats. 2 5 5 The reticulum of dense E C M elements that we demonstrated in the atherosclerotic plaques of W H H L rabbits appeared to be structurally very similar to that shown by Nakamura and colleagues in the rat and very different from the basal lamina of an endothelial cell in a non-diseased regions. The observation of an apparent fragmentation or digestion of the reticulum of dense E C M in the plaques from rabbits exposed to PMio has never been described. This observation raises the question of endothelial cell function in the rabbit 88 Chapter 4: Endothelial Cel l Contacts exposed to PMio . Dysfunction of the endothelium is an important component of 997 9Sft atherosclerotic plaque progression and destabilization. ' Thus it stands to reason that the absence of E C M subtending the endothelium on existing atherosclerotic plaques would increase the plaques' vulnerability to endothelial erosion and potentially plaque rupture. The increase in apparent space beneath endothelial cells to 14.2% in PMio exposed rabbits would further decrease the endothelial anchoring. The space beneath the endothelium is more than likely associated with the large macrophage-derived foam cells lifting the endothelium up off the underlying extracellular matrix, thus the increase in space in PMio exposed rabbits may be attributed to the increase in subendothelial macrophage-derived foam cells in these rabbits. The combined percentage of endothelial intersects with unstable elements (fragmented E C M , macrophage-derived foam cells and space) amounted to 73.9% of all endothelial intersects in the PMio exposed rabbits. These same parameters only amounted to 29.4% of endothelial intersects in the control rabbits. This observation represents a shift in atherosclerotic plaque ultrastructure towards an unstable phenotype following P M i o exposure. The other measured parameters, specifically, contact with macrophages, S M C s , smooth muscle cell derived foam cells and other structures, were similar between the control and the PMio exposed rabbits, an unexpected finding (Figure 4.27). In association with the observed increase in the population of macrophage-derived foam cells contacting the subluminal endothelial plasma membrane following P M i o exposure, a similar increase in macrophages was expected because macrophages are the known precursors of macrophage-derived foam ce l l s . 1 4 7 Furthermore, it was very interesting that there was no change in the populations of S M C s , and S M C derived foam cells in the subendothelial region when comparing the PMio exposed rabbits to the control rabbits. W h y would only the population of macrophage-derived foam cells increase contact with the endothelium? One possible explanation for this observation could be the migration o f already mature macrophage-derived foam cells located in the core of the atherosclerotic plaques up through the fibrous cap and into the subendothelial location where we have demonstrated their accumulation. Another observation that supports this path of migration is that macrophage-derived foam cells containing cholesterol crystals were only observed deep within the cores o f control plaques, 89 Chapter 4: Endothelial Ce l l Contacts yet macrophage-derived foam cells were observed throughout the plaques from rabbits exposed to PMio including a population immediately below the endothelium. Perhaps a population of macrophage-derived foam cells containing cholesterol crystals migrated from the atherosclerotic plaque core up into the region subtending the endothelium in the PMio exposed rabbits. The unknown structures thought to be cells were observed in contact with the endothelium at the same frequency in the PMio exposed rabbits as in the control rabbits. The ultrastructure of these cells was unusual. The intracellular environment was distinguishable from the extracellular environment by the presence o f membranes, yet no organelles, including nuclei, were observed within these cell. The cells all appeared about the same size and shape. A paper by Stary and colleagues 2 5 7 is the only known description of a similar type structure. They propose that these structures are degenerating S M C s and they provided evidence that these structures are extensions of an otherwise healthy appearing S M C . They suggest that as a S M C degenerates, the ends of the cell change first leaving the central portion of the S M C looking unaltered. Although the unknown cell type we observed has a very similar internal composition to the degenerating S M C s documented in that publication, we did not observe that any of these structures were confluent with a S M C . Furthermore, the odd structures were all the same size, and located in the upper regions o f the atherosclerotic plaques. The membrane surrounding them suggests that these structures are not a l ipid artefact of electron microscopy processing; however, their exact nature remains unknown. Regardless of their origin, their small number and apparent lack o f change between atherosclerotic plaques from controls and PMio exposed rabbits suggests that they do not play an important role in endothelial or fibrous cap destabilization. In terms of architecture o f the entire atherosclerotic plaque, many observations in the control W H H L rabbits were made that were supported by the literature. The control plaques were populated by both macrophage related cells and S M C related cells as was expected. The macrophage related cells are typically considered the destabilizing cell type that produce a vast array of cytokines including T N F - a , IL-1 , IL-6, IL-8, IL-10, G M - C S F , and M C P - 1 ; all cytokines involved in atherosclerotic plaque destabilization. 1 3 7 The observation o f the 90 Chapter 4: Endothelial Cell Contacts macrophage-derived foam cells sequestered towards the center of the control atherosclerotic plaques is consistent with a more stable atherosclerotic plaque. In contrast, SMCs are known to be stabilizing cells in atherosclerotic plaques. These cells produce TGF-P which has been shown to not only decrease inflammation, but to also upregulate the production of ECM 1 ^7 components, two important steps required in stable atherosclerotic plaques. The observation of a fibrous cap region populated by SMCs is also consistent with a more fibrous stable phenotype.146 When considered together, the observation of a fibrous cap region populated by SMCs and a lipid core populated by macrophage-derived foam cells is a simple description of an advanced, stable atherosclerotic lesion (type V). That description is a simplification of a complicated process occurring in an atherosclerotic plaque, evidenced by the array of SMC and monocyte type cells throughout the plaque. The altered orientation of the actin rich SMCs between the core and the cap regions of the control atherosclerotic plaques may have been evidence of SMC migration occurring from the media towards the atherosclerotic cap. SMCs are known to migrate146' 2 5 8 as well as proliferate within atherosclerotic plaques, with eventual accumulation in the atherosclerotic plaque cap region.260 Many of the cells observed with altered orientation also had a fragmented or absent basal lamina surrounding the cell, further evidence of migration. Another type of SMC infrequently observed in the control atherosclerotic plaques had a cytoplasm filled with RER and was surrounded by extensive areas of ECM. These SMCs were infrequently observed, though the abundance of RER and their localization in the ECM suggests they play an important role in plaque stability. Cells with a similar appearance to these have been previously observed in atherosclerotic plaques257'261 and there has been the suggestion that this SMC phenotype is responsible for the SMC production of ECM 257 elements and the stabilization of atherosclerotic plaques. The observation and localization of these cells in the upper regions of control atherosclerotic plaques with a fibrous cap corroborates that perception. Small extracellular lipid droplets, like those observed in prior studies,262 were observed in amongst the ECM elements. Collagen was not frequently observed in the atherosclerotic 91 Chapter 4 : E n d o t h e l i a l C e l l Contacts plaques; however , w h e n it was observed, it tended to be associated w i t h the s m a l l extracel lular l i p i d deposits. B o c a n and c o l l e a g u e s 2 6 2 observed a s i m i l a r associat ion between col lagen and extracel lular l i p i d droplets. These extracel lular deposits appeared m u c h smal ler than intracel lular droplets, and were estimated to be about a s ix th o f the size o f intracel lular d r o p l e t s . 2 6 2 T h e observations made here in are i n agreement w i t h that a p p r o x i m a t i o n . E x t r a c e l l u l a r l i p i d droplets were not electron dense. S o m e droplets appeared to have electron dense boundaries, though the major i ty o f s m a l l droplets were apparently u n b o u n d . W h e n large extracel lular droplets were observed, they were t y p i c a l l y associated w i t h cholesterol crystals i n the atherosclerotic plaque core. These droplets resembled the intracel lular droplets suggesting they m a y have resulted from the necrosis o f macrophage-der ived f o a m cells . A l t h o u g h the plaques from the P M i o exposed rabbits contained m a n y o f the same elements observed i n the control plaques, their organizat ion was v e r y different. There was l i p i d accumulat ion; a l though it was not l o c a l i z e d to an atherosclerotic core. There were S M C s o f a l l phenotypes; however , they were observed at a h igher density i n the l o w e r and m i d d l e regions o f the atherosclerotic plaques than the upper region. T h e observat ion o f the greatest importance though has already been discussed above, and that was the p o p u l a t i o n o f macrophage-der ived f o a m cel ls subtending the endothe l ium and a concomitant reduct ion i n endothel ial contact w i t h the r e t i c u l u m o f dense E C M . S M C s are k n o w n producers o f E C M , w h i l e macrophage-der ived f o a m cel ls secrete k n o w n degrading enzymes o f E C M , thus a f ine balance between the t w o populat ions is v e r y important for a stable atherosclerotic plaque. T h e m o r e S M C s and the less macrophage-der ived f o a m cel ls , the m o r e stable the les ion w i l l be. T h e c o m b i n a t i o n o f an increased p o p u l a t i o n o f macrophage-derived f o a m cel ls near the endothe l ium and S M C s i n the l o w e r and m i d d l e regions o f the l e s i o n appears to have resulted i n a deficit o f E C M i n the cap regions o f plaques from P M i o exposed rabbits. In this study w e demonstrated for the first t i m e that w i t h P M i o exposure the endothe l ium is separated from its subtending E C M b y macrophage-der ived f o a m cel ls . In addi t ion, w e have s h o w n here that this process is associated w i t h the fragmentation and degradation o f the r e t i c u l u m o f dense E C M u p o n w h i c h the endothe l ium sat before the arr iva l o f the macrophage-der ived f o a m cel ls . T h i s indicated that these f o a m cel ls do m u c h m o r e than just 92 Chapter 4: Endothelial Cell Contacts insinuate themselves between the plaque endothelium and its underlying reticulum of dense ECM. In fact, our observations strongly suggest that these macrophage-derived foam cells are actively degrading the reticulum of dense ECM, freeing the endothelium and degrading the fibrous caps of previously stable type V atherosclerotic plaques. These observations raise a series of interesting questions. For instance, where do these macrophage-derived foam cells come from? Perhaps there is transmigration from the circulating blood, migration from the core regions of the atherosclerotic plaque or a combination of both sources. Or, what is the role of the truly unique reticulum of dense ECM that has replaced the standard basal lamina of the intima? Is it merely a novel organization of the usual constituents of the basal lamina such as type IV collagen, laminin, fibronectin and perlecan or is it a different type of ECM, perhaps more characteristic of wound healing. To begin to address these issues of the origin of the foam cells we carried out SEM studies to document leukocyte adhesion and migration patterns on control and PMio plaques (Chapter 6). To address the remaining compositional questions, we have undertaken preliminary histochemical analysis (Chapter 5). 93 CHAPTER 5 Composition of Endothelial Basal Lamina 5.1 Introduction T h e E C M matr ix subtending endothel ia l ce l ls , referred to as the basal l am ina , prov ides 9 ^ 9 O'X'X 9 9 8 9 ^ 4 9 ^ 4 9 ^ 6 cr i t ica l support for endothel ia l ce l l morphogenesis , ' pro l i ferat ion, ' m igra t ion , M I 9 ^ 7 9 ^ 8 9 9 Q surv i va l , ' ' and b lood vessel s tabi l izat ion dur ing angiogensis. T h e basal l am ina is composed o f d iverse co l lagen, l am in in and proteoglycan constituents that va ry between 9 9 Q regions o f the vasculature, leve l o f disease burden and stages o f deve lopment and ag ing. ' 263 E v i d e n c e suggests the var ious funct ions o f the E C M m a y be attributed to var ious combinat ions o f the diverse E C M componen ts . 2 2 9 It is c o m m o n l y assumed that the endothel ium is responsib le for the synthesis and depos i t ion o f the basal lamina . In other areas o f the atherosclerot ic les ion the S M C is thought to be p r imar i l y responsib le for the synthesis and deposi t ion o f these same e lemen ts . 1 4 6 W h a t remains u n k n o w n is the regulat ion o f the synthesis and deposi t ion process, par t icu lar ly i n re lat ion to endothel ia l ce l ls . Further to that, the relat ive importance o f spec i f i c E C M components i n support ing endothel ia l ce l l m igra t ion , pro l i ferat ion, and surv iva l is not w e l l understood and often d i f f icu l t to d iscern due to funct ional over lap. There is ev idence o f synergist ic and co-operat ive act ions between combinat ions o f E C M components for a l l the funct ions attributed to the E C M . 2 4 1 There are also suggestions o f s imi lar i t ies o f funct ion between components, w i th va ry ing degrees o f p o t e n c y . 2 6 4 94 Chapter 5: Composition of Endothelial Basal Lamina The term collagen is used to describe a family o f diverse protein molecules that all have a characteristic right-handed triple helix composed of a-chains. Currently 26 types of collagen have been genetically identified, 2 6 5 13 of which are found within the blood vessel w a l l . 2 4 8 Studies suggest the role of collagen in the vasculature includes assisting in the maintenance o f structural integrity, enhancing vessel flexibility and assisting with various cellular events including differentiation, adhesion, migration, proliferation and apoptosis. ' 2 6 5 The combination and relative proportion of collagens within the vessel wall differ with the extent of disease as well as the stage of development and aging. Collagen content increases as atherosclerotic plaques progress. Typically, type I collagen predominates throughout the entire plaque, with detectable levels o f type III and V . These three collagens form fibrils through the tissue performing a more structural support function. Type IV collagen is the most important component of atherosclerotic E C M ' forming a meshwork that acts as a scaffolding for laminins and other E C M components assembly and anchoring. To date there are three heterodimers of type IV collagen documented. 2 6 7 Type IV collagen has a more flexible helical structure than other collagen types, allowing type IV collagen assembly into a meshwork structure that is thin in nascent plaques and increases in thickness as the plaques progress in size and age. 2 4 8 The laminin protein family makes up another important component of the basal lamina of both endothelial cells and smooth muscle cells in atherosclerotic plaques. More than 15 different members of this family have been identified and each is composed of various combinations of three subunits, spliced together typically in a cross-shaped pattern. 2 6 7 Laminins are an important element in the basal lamina, second only to type I V collagen and they play a vital role in atherosclerotic cap stabilization. 2 6 7 Individual laminin protein subunits are produced by most cells, including endothelial ce l l s . 2 6 8 The individual subunits of the laminin molecule then self assemble in the extracellular environment with the help of 9fi"7 Of\Q specific cellular anchoring points. ' The current perception in the literature is that regulation of laminin content in basal lamina occurs at the level of synthesis and degradation of the individual laminin subunits in cells rather than at the extracellular laminin assembly s tep . 2 6 3 ' 2 6 9 95 Chapter 5: Composition of Endothelial Basal Lamina The unique 3-dimensional organization of the reticulum of dense E C M subtending the endothelium in the location where one would expect to find a basal lamina (Chapter 4) has not been previously reported with the possible exception of the renal corpuscles of diabetics which have an uncharacterized, thickened basement membrane structure observed surrounding the glomerular arteries. 2 7 0 The composition and organization o f the dense reticulum observed in atherosclerotic plaques in this study is o f special interest to determine how it compares to the structural elements in non-diseased basal lamina because of its presumed role in support of the endothelium and its apparent fragmentation in the PMio exposed rabbit plaques. Because o f their known role in basal lamina composition and function, the presence or absence o f type IV collagen and laminins in the reticulum of dense E C M o f the cap regions o f the atherosclerotic plaques was o f particular interest. 5.2 Aim To determine whether the subendothelial reticulum of dense E C M of atherosclerotic caps is histochemically similar to the known composition of basal lamina of normal artery and therefore able to perform similar functions. 5.3 Methodology Exposure protocols and tissue processing protocols were outlined in Chapter 2. The following methodology was applied to formalin fixed, paraffin embedded tissue. 5.3.1 Microanatomical Staining 12 Paraffin blocks o f atherosclerotic aorta from 10 separate W H H L rabbits were sectioned at 5 pm and stained with hematoxylin / eosin, periodic acid-Schiff (PAS) / hematoxylin, alcian blue - P A S (pH 2.5), and a silver based reticulin stain. Standard hematoxylin and eosin (H & E) staining was performed on deparaffinized, hydrated slides. Slides were stained with hematoxylin for 10 minutes, rinsed with tap water for 5 minutes and then placed in 1% HC1 in 70% ethanol for 10-15 seconds (1 to 2 dips). Slides were then rinsed in tap water before being placed in a weak ammonia solution for 90 seconds 96 Chapter 5: Composition of Endothelial Basal Lamina and then washed in running tap water for 10 minutes. Following washing, slides were placed in 80% ethyl alcohol for 2 minutes and then counterstained with eosin for 2 minutes. Slides were then dehydrated, and cover slipped. Eosin is a cytoplasmic stain and hematoxylin is a nuclear stain, thus the cytoplasm of cells appeared reddish and the nuclei of cells appeared blue following H & E staining. This stain allowed us to identify the endothelial cells lining the arterial lumen when the endothelium was present. The P A S staining procedure oxidizes free hydroxyl groups to aldehydes, which are then demonstrated by the Schiff reagent allowing the identification of simple polysaccharides, 971 neutral mucosubstances and basement membranes. For the P A S - hematoxylin staining, deparaffinized slides were placed for 10 minutes in a 0.5% periodic acid solution, rinsed three times in distilled water and stained for 15 minutes in Schiff reagent followed by a second rinse in running tap water for 5 - 10 minutes. Slides were counterstained with hematoxylin for 1 minute, rinsed again, dried and cover slipped. The P A S positive materials in an atherosclerotic cap include basement membranes, collagen, and reticular fibres. These materials w i l l stain magenta using the P A S procedure, while all cell nuclei w i l l be stained blue by the hematoxylin. The Alc ian blue - P A S staining procedure involved staining the slides for 30 minutes in alcian blue solution in 3% acetic acid solution, and washing under running water for 2 minutes. Slides were briefly rinsed with 3% acetic acid, placed for 20 minutes in a 0.5% periodic acid solution, and rinsed three times in distilled water. Slides were then stained for 20 minutes in Schiff reagent prior to a 5 - 10 minute rinse in running tap water. Slides were dehydrated, cleared and cover slipped. The alcian blue in a 3% acetic acid solution stains the negative charged areas of both sulphated and carboxylated acid mucopolysaccharides as well as sulphated and carboxylated glycoproteins blue . 2 7 1 In an atherosclerotic cap dermatan sulphate and chondroitin sulphates, the polysaccharide component of most major proteoglycans including perlecan, 2 7 2 w i l l react with the alcian blue stain. The P A S positive materials throughout atherosclerotic plaques include basement membranes, collagen, and reticular fibres and they should stain magenta. 2 7 1 Areas with both elements w i l l appear pu rp l e 2 7 1 97 Chapter 5: Composition of Endothelial Basal Lamina The Gomori reticulin stain is a silver based stain that turns reticular fibres black. Deparaffinized, hydrated slides were oxidized in 0.5% potassium permanganate for 1 minute then rinsed well in tap water. Slides were differentiated for 1 minute using 2% potassium metabisulfite, and then again rinsed in tap water. Slides were then sensitized for 1 minute in 2% ferric ammonium sulphate, rinsed well in tap water and put through two washes in distilled water, each lasting 1 minute. Slides were treated with ammoniacal silver solution for 1 minute, rinsed in distilled water and reduced in 20% formalin for 2 - 3 minutes. Slides were washed for 3 minutes in running tap water prior to toning in 0.2% gold chloride for 12 minutes. Slides were rinsed in distilled water and then treated with 2% potassium metabisulfite for 1 minute, fixed in 2% thiosulfate for 1 minute and washed well in running tap water. Slides were dehydrated, dried, cleared and coverslipped. A l l slides were examined for staining using a Nikon Labophot-2 Light Microscope and photographed on the Nikon Eclipse 50i. Scale bars were added and images were cropped in Photoshop. 5.3.2 Immunohistochemical Staining Immunohistochemistry staining for type IV collagen and laminin was performed on 5 pm sections of the same rabbit aortas. Our primary positive controls were human prostate, and rabbit lung, the latter being a positive control specific for the rabbit tissue. Two antibodies for type IV collagen were tested, mouse anti-human Collagen IV (Serotec) and M 3 F 7 (University of Iowa, Hybridoma Bank) and two antibodies for laminin were tested, Lam-89 (Novus Biologicals) and 2E8 (University of Iowa, Hybridoma Bank). Several different antigen retrieval protocols were attempted on the deparaffinized slides, including 0.1% trypsin for 20 minutes, 1% trypsin for 20 minutes, Proteinase K for 10 minutes, Pepsin in 0.5N glacial acetic acid at 37°C for 2 hours, citrate buffer (pH 6) in the autoclave, T B S buffer (pH 9) in the autoclave, citrate buffer (pH 6) in the microwave, and T B S buffer (pH 9) in the microwave. 98 Chapter 5: Composition of Endothelial Basal Lamina Following the antigen retrieval protocol, slides were rinsed twice with T B S (pH 7.6) for five minutes and then blocked for 20 minutes using Protein Block (DakoCytomation). Fluid was tapped off slides prior to 50 ul of the primary antibody being added for 1 hour. For each separate antigen retrieval protocol, titrations of the primary antibody were done at a 1/50, 1/100 and 1/500 ratio of antibody to a solution of T B S buffer and B S A . Following incubation with primary antibody, slides were rinsed twice with T B S (pH 7.6) for five minutes, and the secondary antibody, polyclonal rabbit anti-mouse immunoglobulins (DakoCytomation), was added at a dilution of 1/20 for 30 minutes. Slides were then rinsed twice with T B S (pH 7.6) for five minutes, and the tertiary antibody, A P A A P mouse monoclonal (DakoCytomation), was added at a dilution of 1/50 for 30 minutes. Slides were again rinsed twice with T B S (pH 7.6) for five minutes prior to substrate addition for 20 minutes. Slides were rinsed with water, counterstained with hematoxylin and then well rinsed in water before a quick graded dehydration protocol. Once dried, slides were cover slipped and analyzed. A l l slides were examined for staining using a Nikon Labophot-2 Light Microscope and photographed on the Nikon Eclipse 50i. Scale bars were added and images were cropped in Photoshop. 5.4 Results 5.4.1 Microanatomical Staining H & E staining of atherosclerotic plaques from both control and PMio exposed rabbits showed that the endothelium was largely absent from these arteries leaving a continuous layer of dense E C M along the luminal surface of the control atherosclerotic plaque and a discontinuous layer of dense E C M along the luminal surface of the atherosclerotic plaques from PMio exposed W H H L rabbits (Figures 5.1a and 5.2a). This observation was confirmed by T E M analysis of 4% paraformaldehyde fixed atherosclerotic plaques processed for immunogold labelling (Figure 5.3). P A S - hematoxylin staining exhibited pronounced staining within the fibrous cap region and a continuous layer of carbohydrate moieties along the plaque surface of atherosclerotic plaques from control animals (Figure 5.1b). The thickened basement membrane surrounding 99 Chapter 5: Composition of Endothelial Basal Lamina S M C s was clearly stained as was" material in the intercellular spaces. Staining appeared more pronounced in the upper regions of the atherosclerotic plaques, over the plaque core accumulations of macrophage-derived foam cells in both control and exposed groups. In contrast to control plaques, the atherosclerotic plaques from PMio exposed rabbits exhibited a discontinuous layer of staining for carbohydrate moieties as well as some staining of cells in the upper regions of the plaque. There was not a clear thickening of P A S positive material surrounding cells, and there was minimal to no staining of the intercellular spaces. T E M analysis showed that while the endothelium had been lost, the reticulum of dense E C M observed in Chapter 4 remained (Figure 5.3). Alc ian blue - P A S staining identified a thin layer of alcian blue positive material along the luminal border of the plaques localized in an area believed to be subtending the endothelium. This layer was more continuous in the plaques from control rabbits, where as the plaques from PMio exposed rabbits had clear, interruptions in the layer, typically associated with regions were the endothelium was difficult to identify or absent (Figure 5.1c versus 5.2c). Plaques from control rabbits had more regions rich in dermatan sulphate and chondroitin sulphate containing proteoglycans than did plaques from PMio exposed rabbits. The basement membrane surrounding S M C s clearly stained magenta in the both control and PMio , though more prominently in the control atherosclerotic plaques. The reticulin stain was negative for reticular fibres in plaques from both the control and the PMio rabbits. 5.4.2 Immunohistochemical Staining Within positive control tissue (human prostate) the basal lamina surrounding S M C s and within nerve bundles was clearly outlined by the staining procedure (Figure 5.4). The staining of rabbit lung and aortic tissue for laminin and type IV collagen was unsuccessful. Stained tissue appeared identical to control IgG stained tissue, regardless o f the many antigen retrieval protocols used (Figure 5.5). The exception to this was staining in cartilaginous airways of the lung where staining occurred in the golgi region above the nuclei of epithelial cells (Figure 5.6). 100 Chapter 5: Composition of Endothelial Basal Lamina • t Figure 5.1. Atherosclerotic plaque from control W H H L rabbits, a) H & E stain identifies endothelial nuclei (white arrows) along only a portion of the surface o f the atherosclerotic plaque and many S M C s in parallel orientation to the plaque endothelium, b) P A S stain identifies a continuous layer o f carbohydrate moieties along the plaque surface (arrowheads). The E C M surrounding S M C s throughout the plaque (black arrows) and a dense cap rich in basement membranes and collagen are clearly stained, c) Alcian blue - P A S stain verifies the layer o f carbohydrate moieties along the plaque surface (arrowheads), extensive amounts of proteoglycans throughout the atherosclerotic plaque and P A S positive material surrounding S M C s distributed throughout the plaque (black arrows), d) Reticulin stain was negative for reticular fibres. A l l scale bars = 50 pm. 101 Chapter 5: Composition of Endothelial Basal Lamina a. * » b. • flfl d. Figure 5.2. Atherosclerotic plaque from PMio exposed W H H L rabbits, a) H & E stain identifies endothelial nuclei (white arrows) sporadically placed along the surface o f the atherosclerotic plaque. The upper regions of the plaque appear very cellular though the cell type is unknown, b) P A S stain outlines the discontinuous layer of simple polysaccharides and neutral mucosubstances along the luminal surface of the plaque (arrowheads), surrounding S M C s (black arrows), and throughout the plaque, c) Alc ian blue - P A S stain verifies the discontinuous layer o f acidic and sulphated carbohydrates along the luminal surface of the plaque (arrowheads), surrounding S M C s (black arrows), and throughout the plaque, d) Reticulin stain was negative for reticular fibres. A l l scale bars = 50 pm. 102 Chapter 5 : Composition of Endothelial Basal Lamina 3034 — 2 - 7 — B r d U - 8 K - 3 , l . t i f P r i n t Mag: 8830x g 200 mm 2 » i c r o M \ c q u i r e d Dec 1, 2004 a t 3:17 PM HV= SOlcV fEM Mode: HC-ZOOM D i r e c t Mag: 7000x Figure 5.3. Absent endothelium in 4% paraformaldehyde fixed atherosclerotic plaque. The reticulum of dense E C M (white arrows) along the luminal surface of an atherosclerotic plaque that was fixed with formalin for immunogold labelling. Micrograph reproduced with permission from Dr. Ali Behzad. 103 Chapter 5: Composition o f Endothelial Basal Lamina Figure 5.4. Positive staining for laminin and type IV collagen in the human prostate, a) IgG control in human prostate. Scale bar = 1 0 0 pm. b) Inset o f panel a. N o staining was detected in the control tissue. Scale bar = 50 pm. c) Positive laminin staining in human prostate. Scale bar =100 pm. d) Inset of panel c. Staining is localized to areas surrounding cells known to be rich in basal lamina. Scale bar = 50 pm. e) Type I V collagen staining in human prostate. Scale bar = 100 pm. f) Inset of panel e. Localized staining for collagen in regions rich in basal lamina. Scale bar = 50 pm. 104 Chapter 5: Composition of Endothelial Basal Lamina Figure 5.5. Immunohistochemistry analysis for laminin and type IV collagen in the W H H L rabbit, a) IgG control in rabbit lung, b) IgG control in rabbit aorta, c) Negative laminin staining in rabbit lung, d) Negative laminin staining in rabbit aorta, e) Negative type IV collagen staining in rabbit lung, f) Negative type I V collagen staining in rabbit aorta. A l l scale bars — 100 pm. 105 Chapter 5: Composition o f Endothelial Basal Lamina Figure 5.6. Staining in the golgi region of W H H L rabbit lung epithelial cells, a) IgG control in rabbit lung. Scale bar = 100 pm. b) Inset of panel a. N o staining observed in IgG control. Scale bar = 50 pm. c) Staining for laminin in rabbit lung. Scale bar = 100 pm. d) Inset of panel c. Clear staining of the golgi region of epithelial cells for laminin (arrows). Scale bar = 50 pm. e) Staining for type I V collagen in rabbit lung. Scale bar = 100 pm. f) Inset o f panel e. Clear staining o f the golgi region for type I V collagen in epithelial cells (arrows). Scale bar = 50 pm. 106 Chapter 5: Composition of Endothelial Basal Lamina 5.5 Discussion The results confirm the presence of a continuous layer o f carbohydrate moieties along the plaque surface from control W H H L rabbits. Furthermore, thickened layers of acid and sulphated carbohydrate moieties surrounding S M C s and in intercellular spaces in atherosclerotic plaques were observed throughout control samples. In the atherosclerotic plaques of W H H L rabbits exposed to PMio a discontinuous surface layer of carbohydrate moieties and an absence of intercellular staining was observed. Immunohistochemistry staining to determine whether the material observed subtending the endothelium contained laminin and type IV collagen proved unsuccessful with the antibodies available to us. The basic microanatomical and histochemical staining procedures used in these studies were capable of demonstrating the presence of proteoglycans, basal lamina, collagen, and reticular fibres in the atherosclerotic plaques. Although the endothelium was largely absent from these arteries, a layer of acid and / or sulphated proteoglycan containing material was demonstrated on the luminal surface of control atherosclerotic plaques. Dr. Behzad's T E M observation from these same arteries confirmed that this layer is mostly the reticulum of dense E C M described in Chapter 4. L M staining protocols identify the presence or absence o f the elements but can not resolve the detail o f their 2- or 3-dimensional organization to the extent reported in the T E M observations of Chapter 4. The fragmentation of the reticulum of dense E C M subtending the endothelium observed in Chapter 4 in plaques from PMio exposed rabbits would lead one to predict that histochemical staining for acid and / or sulphated proteoglycan containing materials provides evidence o f their presence rather than their 2- or 3-dimensional organization. The observations presented herein o f E C M elements in the atherosclerotic plaques from PMio exposed rabbits do in fact suggest that the surface layer of E C M in PMio plaques is disrupted or reduced in P A S and Alc ian blue - P A S stained material. These observations support the observation in Chapter 4 suggesting that macrophage-derived foam cell accumulation under plaque endothelium is accompanied by damage and or reduction of this special basal lamina like material. Although immunohistochemical staining worked in human control tissues, we were unable to achieve immunostaining of the basal lamina in endothelial cells of the rabbit aorta, and of 107 Chapter 5: Composition of Endothelial Basal Lamina epithelial and endothelial cells of the rabbit lung. Both endothelial cells and epithelial cells 273 are known to be subtended by basal lamina material, rich in type IV collagen and laminin. ' 274 W e were surprised to see staining of the golgi apparatus of epithelial cells l ining cartilaginous airways. Potentially protein subunits of laminin and type I V collagen in the golgi apparatus reacted with their respective primary antibodies. Laminin is a molecule that is composed of protein subunits that are translated in the R E R of cells, endothelial and epithelial cells included, glycosylated and sulphated in the golgi apparatus and then released as individual subunits into the extracellular environment. 2 6 8 In the extracellular environment the laminin subunits self assemble. ' Type IV collagen is also produced within the cell through the same synthetic apparatus and then released into the extracellular environment. Wi th the exception of the Laminin Lam-89 antibody, all the antibodies had not been previously tested in rabbits. Though the collagens and the laminins are highly conserved protein structures, 2 7 5 it is possible that a slight change to the epitope between the human and the rabbit may render the antibody ineffective in the rabbit. The golgi region of epithelial cells in the rabbit lung stained positively for both laminin and type IV collagen. We suggest that this could be due to the unassembled state o f the precursor proteins in the golgi apparatus allowing them to cross react with the human targeted antibodies, whereas following assembly o f the laminin and type IV collagen in the rabbit basal lamina, the cross reacting epitope may no longer be available. Finally, it may be relevant that even in human prostate material, the basal lamina subtending the glandular epithelial cells did not stain with either of these antibodies. Conventional monoclonal antibody production begins with the inoculation o f an animal with the antigen o f interest. Most antibodies are produced in rabbits and targeted at protein epitopes in other species, such as the mouse or the human. This has led to a scarcity o f antibodies that can be used to investigate the presence of protein epitopes in rabbits. This was the challenge we faced in the identification of laminin and type IV collagen in the aorta o f W H H L rabbits. The antibodies chosen were done from a very limited pool of antibodies available that had not been raised in rabbit and that targeted our proteins o f interest. In the 108 Chapter 5: Composition of Endothelial Basal Lamina future, a study using type IV collagen and laminin targeted antibodies raised against the rabbit would be very useful in determining the composition of the dense E C M material. Furthermore, perlecan and versican targeted antibodies, which are the two most prevalent on(\ onQ proteoglycans in the basement membrane o f atherosclerotic plaques, " could be included to see i f these proteoglycans were components of the dense E C M material. The role of proteoglycans as a scaffolding for cellular attachment and vessel wall stabilization has been recognized in the last decade, as has the potential prothrombotic function of proteoglycans in the event of a plaque rupture. Though the precise composition of the reticulum o f dense E C M observed remains to be elucidated, information was gained regarding the composition of the reticulum of dense E C M within the cap regions of atherosclerotic plaques as well as the degradation of this material following PMio exposure. In this study we have demonstrated by histochemical techniques that the reticulum of dense E C M described in Chapter 4 at least contains acid and/or sulphated carbohydrate moieties as would be the case for basal lamina. This light microscopic staining was the reticulum of dense E C M seen ultrastructurally. Wi th the accumulation of macrophage-derived foam cells under PMio treated endothelium (Chapter 3) and the alteration and degradation of the E C M subtending the endothelium (Chapter 4 and 5), the effects of these alterations on the plaque endothelium remained to be determined. In the next Chapter, the scanning electron microscope was used to accomplish this. 109 CHAPTER 6 Surface Morphology of Atherosclerotic Plaques 6.1 Introduction The arterial endothelium is a squamous cell monolayer at the interface between circulating blood and all surrounding tissues. In a non-diseased artery, the endothelial cells are spindled-osfi 99.9 — shaped with their long axis oriented in the direction of blood flow. " The nucleus o f the cell lies flat, producing a low protrusion on the luminal surface of the endothelial c e l l . 1 4 5 Scanning electron microscopy ( S E M ) reveals a smooth surface with cytoplasmic flaps o f adjacent cells overlapping to create a complex barrier. Transmission electron microscopy ( T E M ) shows that adjacent endothelial cells are held together by intercellular junctional complexes that typically include tight junctions, adherens type junctions and gap-junctions near the luminal surface. 1 4 5 ' 2 8 3 S E M observations show clearly that although endothelial cell shape changes in areas of bifurcation, the cells still remain aligned with the direction o f blood 9R9 flow. A s vessels narrow and hemodynamic forces change, endothelial cell morphology 9R^ changes; cells appear more elongated, microfilaments and intermediate filaments are increased, and the E C M thins, all effects of blood flow and shear stress on the endothelial surface . 1 4 1 ' 1 4 3 The topography o f the endothelium changes over atherosclerotic plaques. In response to atherosclerotic risk factors, particularly circulating L D L and free radicals, the expression o f I C A M - 1 , 1 3 2 V C A M - 1 , 1 3 2 ' 1 3 3 E-selectin and P-selectin 1 3 2 ' 1 3 4 increase on the endothelial surface. Leukocytes, particularly those activated by circulating L D L and cytokines, begin rolling and then tether to the adhesion molecules on the endothelial surface (Figure 6.1). 1 2 8 ' 110 Chapter 6: Surface Morphology o f Atherosclerotic Plaques 1 3 5 Following tethering, leukocytes may establish firm adhesion to the surface of the endothelium and crawl along the surface of the endothelium to a location where transmigration across the endothelium and into the blood vessel wall can occur; often transmigration occurs at tricellular corners . 2 8 4 , 2 8 5 Figure 6.1. An illustration of leukocyte rolling, tethering, adhesion and migration. Figure adapted from Diacovo 1996 2 8 6 and Cines 1998. 2 3 0 Transmigration o f leukocytes associated with the growth of atherosclerotic plaques has been shown to occur primarily at the plaque shoulders. 2 8 7 Monocytes are the principal leukocyte which transmigrate into the plaque at the shoulders where upon they become activated macrophages scavenging lipids and ultimately evolving into lipid filled macrophage-derived foam c e l l s . 1 4 7 , 2 8 7 " 2 9 1 Thus monocyte attachment and transmigration at the shoulder regions of the plaques and their activities within the plaque at the shoulders are believed to be responsible for the inherently unstable nature of plaque shoulders. 1 5 9 , 1 6 7 Atherosclerotic plaque rupture typically occurs at the shoulder regions in association with monocyte Rupture of the plaque leads to thrombosis formation and acute infiltration 220, 292 cardiovascular events 293 However, our L M and T E M observations in PMio exposed rabbit plaques suggested that leukocyte activity is not primarily occurring at the plaque shoulder, but rather that the central cap region o f the plaques is where macrophage-derived foam cells accumulate and the E C M subtending the endothelium fragments (Chapters 3 and 4). These are features of instability not typically associated with the central regions of atherosclerotic plaques. Therefore, the 111 Chapter 6: Surface Morphology of Atherosclerotic Plaques focus o f this investigation has been to assess endothelial integrity, topography and leukocyte trafficking over atherosclerotic plaque cores and at atherosclerotic plaque shoulders of control and PMio exposed W H H L rabbits using the high resolution Hitachi S4700 field emission gun S E M . 6.2 Aim To define any observed differences in endothelial topography and document leukocyte trafficking on plaque surfaces of PMio exposed and control W H H L rabbits. 6.3 Methodology S E M observations were made on the abdominal aortas harvested using the perfusion fixation protocol. Samples from 5 control and 5 PMio exposed W H H L rabbits were randomly chosen during tissue processing (please refer to Chapter 2 for full details). Once samples had been processed and prepared for S E M analysis, all samples were analyzed on a Hitachi S4700 field emission gun S E M . Emphasis was placed on atherosclerotic plaque core and shoulder regions, though some observations were also made on non-diseased areas of the arterial wall . Representative micrographs were captured from all investigated areas. A n approximation of the areas analyzed using the S E M was obtained by measuring the length and the width o f all the pieces o f abdominal aorta under a dissecting microscope. The total area investigated in the PMio samples was 207 m m 2 with 7 large atherosclerotic plaques visible. In the control samples the total area investigated was 216 m m 2 with 9 atherosclerotic plaques visible. N o individual segment o f abdominal aorta had more than 1 atherosclerotic plaque though some rabbits had multiple atherosclerotic plaques from different regions within the abdominal aorta. , 6.4 Results Qualitative S E M analysis of the arterial wall revealed squamous endothelium in non diseased areas (Figure 6.2) in both the control and the PMio exposed W H H L rabbits. Flat cytoplasmic extensions reached over the surface of neighbouring cells, creating a ruffled appearance to the endothelial cell borders. The long axis of endothelial cells was orientated in the direction 112 Chapter 6: Surface Morphology of Atherosclerotic Plaques of blood flow, except at bifurcations where endothelial cell shape and orientation were altered somewhat (Figure 6.3a and 6.3b). The surfaces o f the non-plaque endothelial cells were typically smooth, without surface projections of any type visible. A t the upper limit o f resolution of the S E M , 10 - 15 nm caveolar entrances were visible on the endothelial surfaces o f non-diseased blood vessel wall , and the endothelial surfaces of atherosclerotic plaques in both control and PMio exposed rabbits (Figure 6.4). Figure 6.2. Surfaces of non plaque endothelium, a) Spindle shaped squamous endothelial cells have many interdigitating cytoplasmic flaps (arrows) overlapping at cell borders in a non-diseased area of a control arterial wall, b) Non-diseased regions from a PMio exposed rabbit are equivalent to non-diseased regions observed in control rabbits. Figure 6.3. Endothelial cell orientation at an arterial bifurcation, a) The unique orientation of the endothelium at an arterial bifurcation, b) Tracing o f endothelial cell borders in panel a to assist in border identification. 113 Chapter 6: Surface Morphology o f Atherosclerotic Plaques Figure 6.4. Caveolae on the surface of atherosclerotic plaque endothelium, a) The border between two endothelial cells (arrows) over a control atherosclerotic plaque, b) Endothelial caveolae (arrowheads) in the luminal plasma membrane. The atherosclerotic plaques in both control and PMio exposed rabbit aortas were easily identified by S E M on the arterial walls projecting into the arterial lumen (Figure 6.5). Plaques frequently extended upstream of arterial bifurcations (Figure 6.5a). The irregularly shaped contours of the shoulder regions of the atherosclerotic plaques were not uniform (Figure 6.5a and 6.5c). Trailing edges were apparently more regular and more gently sloped than the leading edge o f the plaque (Figure 6.5b versus 6.5c). When viewed in section at the edge of a piece of tissue, the thickening of the intima o f the plaque was clearly visible projecting into the vessel lumen (Figure 6.6a). In some cases the plaque separated from the media of the arterial wal l during the process of critical point drying (Figure 6.6a) presumably due to shrinkage. In addition to their elevation, plaques could easily be identified on their cut edges by the contents o f the atherosclerotic plaque which differed greatly from the regularly layered organization of the media. In contrast to S M C s separated by layers of elastic lamina, investigation of the cut edge of atherosclerotic plaques revealed the disordered accumulations of fibrous material, and pits about 2 to 3 pm in diameter in cells representing the fat droplets within the foam cells (Figure 6.6b and 6.6c). Although the endothelium over the core region of control atherosclerotic plaques appeared squamous, the topography was more uneven than areas of endothelium from non-diseased arterial wall . This gave the core surface of control atherosclerotic plaques a rumpled or rolling appearance. Small areas of lateral endothelial cell separation were observed (Figure 6.7c) and the endothelial borders had a more irregular, roughened appearance when 114 Chapter 6: Surface Morphology o f Atherosclerotic Plaques Figure 6.5. Topography of an atherosclerotic plaque, a) A n atherosclerotic plaque located upstream of an arterial bifurcation (*). b) The trailing edge of the atherosclerotic plaque with a regular border, c) The leading edge of the atherosclerotic plaque with an irregular border. 115 Chapter 6: Surface Morphology o f Atherosclerotic Plaques Figure 6.6. Cross section of an atherosclerotic plaque from a control W H H L rabbit. a) A low magnification o f an atherosclerotic plaque (A) and the underlying media (*). b) Higher magnification of the atherosclerotic plaque. E C M elements are visible and so are l ipid droplets, c) Detail o f the atherosclerotic plaque. Small pits located in among the matrix (arrowhead) and a structure that is potentially a cell edge (arrow) is visible. 116 Chapter 6: Surface Morphology o f Atherosclerotic Plaques Figure 6.7. Control atherosclerotic plaque endothelium, a) Irregular topography o f the core region of an atherosclerotic plaque. Areas protrude into the lumen more than expected for an endothelial nucleus (•). b) The endothelium from the core region is intact, some cytoplasmic flaps are visible (arrows), though the cell borders appear a bit ruffled, c) Potential cell separation (arrowheads) in the endothelium over the core o f the control atherosclerotic plaque, d) Very uneven cell margins and a small area o f separation visible on the endothelium of a plaque shoulder, e) Area of leukocyte activity with two attached cells (*) at the shoulder of a control plaque, f) A visible cell (*) in the endothelial opening of the shoulder region o f a control plaque. 117 Chapter 6: Surface Morphology o f Atherosclerotic Plaques compared to non-diseased areas of the vessel wall. On the shoulder regions, the endothelium of the control atherosclerotic plaques was irregular with large sites o f intercellular gaps associated with attached and transmigrating leukocytes (Figure 6.7e and 6.7f). Observations of atherosclerotic plaques from PMio exposed rabbits revealed extensive leukocyte adhesion and transmigration over the plaque cores (Figure 6.8 and Figure 6.9). Areas of multiple cell adhesion, with associated platelets and red blood cells were observed (Figure 6.9). The endothelium was frequently observed protruding into the arterial lumen. These protrusions were about the size and shape of leukocytes observed adhering to the Figure 6.8. Atherosclerotic plaque endothelium from a PMio exposed rabbit, a) Area of leukocyte adhesion (*) and transmigration over an atherosclerotic core with areas o f luminal endothelial protrusion (arrows), b) Focal endothelial bulge over the plaque core with two leukocytes (*) attached and/or transmigrating at an endothelial cell border (arrowhead), c) Area of leukocyte adhesion and transmigration with a clear intercellular gap in the endothelium (white arrow), d) Inset of panel c. Contents o f the atherosclerotic plaque are visible through the gap (white arrow). Adjacent cell (•) may be immigrating into the plaque. 118 Chapter 6: Surface Morphology of Atherosclerotic Plaques Figure 6.9. A leukocyte cluster over the core of an atherosclerotic plaque from a PMio exposed rabbit, a) A group o f leukocytes attached to and transmigrating at the endothelial surface. The black and white arrows point to the same two red blood cells (RBC) throughout the panels, b) Detailed image of the lower section of the leukocyte cluster and surface R B C (arrows), c) Detailed image o f the upper section of the leukocyte cluster, d) A n inset from panel c of adherent platelets (arrowheads) attached to both the endothelium and the edge of a leukocyte, e) A detailed image o f the attached R B C (black arrow). Platelets (arrowheads) are attached to the cluster o f leukocytes, f) A detailed image of the attached red blood cell (white arrow). Platelets and fibrin resting in the dip of the R B C as the R B C sits on the leukocytes. 119 Chapter 6: Surface Morphology o f Atherosclerotic Plaques luminal endothelial surface. B y T E M large accumulations of macrophage-derived foam cells under the endothelium over both shoulder and core regions of atherosclerotic plaques were observed (Figure 6.10). The subendothelial macrophage-derived foam cells were responsible for the protrusion o f the endothelium into the arterial lumen that easily exceeded the heights of endothelial nuclei (Figure 6.11). Figure 6.10. Endothelial cells protruding into the lumen due to subendothelial macrophage-derived foam cells, a) A macrophage-derived foam cell undergoing endothelial transmigration at a plaque shoulder and the endothelium is being pushed outwards by subtending macrophage-derived foam cells. The internal elastic lamina in an unaffected region of the blood vessel wall is visible (arrows) below the bulging endothelium. Scale bar = 5 um. b) Macrophage-derived foam cells distend the endothelium into the arterial lumen over the core of an atherosclerotic plaque. Scale bar = 5 pm. In addition to leukocyte attachment and subendothelial accumulation, transmigration was observed at both bicellular (Figure 6.8b) and tricellular corners o f endothelial cells (Figure 6.12). Directionality of a transmigrating leukocyte can be difficult to determine from fixed tissue, but specific interactions between endothelial cells and migrating cells provide clues. A s a cell immigrates into the blood vessel wall , it may push the endothelial cell down into the atherosclerotic plaque in such a fashion that the edges of the endothelial cell are no longer visible (Figure 6.13a). B y contrast, i f a cell emigrates out of the wall into the blood vessel lumen, it may pull the endothelial cell outwards into the lumen of the blood vessel at 120 Chapter 6: Surface Morphology o f Atherosclerotic Plaques Figure 6.11. Macrophage-derived foam cell lifting the endothelium up into the blood vessel lumen. The endothelial border is outlined with white arrows and an * marks the macrophage-derived foam cell. !,SkV 12 7mm x7 OC* SEIMl 7/18/05 I I I I I 5 OOum S4700 2 5kV 12 7mm x.30 Ok SEIMl 7/18/05 1 OOum Figure 6.12. Leukocyte trail, a) A transmigrating leukocyte (*) with a neighbouring gap between endothelial cells (black arrow) and surrounded by material on the endothelial surface (white arrows), b) Interendothelial gap and surrounding deposits of the material on the endothelial surface. 121 Chapter 6: Surface Morphology of Atherosclerotic Plaques sites of focal adhesion (Figure 6.13b). 2 9 4 S E M analysis revealed endothelial filopodia extending up the sides of cells transmigrating at the plaque surfaces. Specific points of contact on the migrating cell suggest sites o f focal adhesions between the endothelial cell and the migrating cell. We believe the observations of endothelial filopodia extensions up the sides of transmigrating cells indicate that these cells are actually emigrating out o f the vessel wall into the lumen. Over the atherosclerotic core of PMio rabbits, many of these small endothelial filopodia were observed extending up the sides of transmigrating cells (Figure 6.14a to 6.14d) suggesting emigration. Striking evidence for the emigration of macrophage-derived foam cells was the observation of such a cell carrying fragments of the reticulum of dense E C M elements from the subendothelial region into the arterial lumen (Figure 6.15). A n absence of endothelial filopodia on the side of the migrating cell (Figures 6.8, 6.14e and 6.14f) does not immediately mean the cell is immigrating into the atherosclerotic plaque from the lumen; rather, the observation prevents a conclusive statement regarding the direction of migration. S E M observations suggestive of immigrating cells were infrequent in both control and PMio exposed groups. The most probable S E M observations of possible cell immigration are Figures 6.8, 6.14e and 6.14f. More difficult to find and discern were T E M observations o f cells immigrating from the circulation into the atherosclerotic lesion. Figure 6.16 is the only T E M observation of a cell that may be immigrating across the endothelium into the blood vessel wall . In control rabbits, leukocyte transmigration was mostly confined to the atherosclerotic plaque shoulders. In PMio exposed rabbits the majority of transmigration was observed over the atherosclerotic plaque core and appeared to be emigration from the atherosclerotic plaque into the arterial lumen. In addition to leukocyte transmigration, areas of endothelial desquamation were also observed (Figure 6.17a) and these areas usually exhibited platelet adhesion to the exposed E C M (Figures 6.17b, and 6.17c). In contrast to the normal large artery endothelial basal lamina which is organized as a sheet with apertures, the exposed E C M in areas of endothelial desquamation on plaques was clearly organized as a reticulum o f dense E C M (Figure 6.17d) as demonstrated by the T E M data in Chapter 4. 122 Chapter 6: Surface Morphology o f Atherosclerotic Plaques Blood vessel wall b) Emigration out of the blood vessel wall Figure 6.13. An illustration of leukocyte immigration and emigration, a) Immigration into the blood vessel wall is characterized by the migrating cell pushing the endothelial cell into the vessel wall , obscuring the borders of the cell, b) Emigration out of the atherosclerotic plaque is characterized by the migrating cell pushing and pulling the endothelium outwards into the lumen of the blood vessel wall . Endothelial filopodia are visible extending up the sides of the migration cell. 123 Chapter 6: Surface Morphology o f Atherosclerotic Plaques S4700 2 5kV 12 Omm x3 001 SEIUI 8/5/05 S4700 2 51V 12 1mm x4 50k SEfUl 8/5/05 Figure 6.14. Migrating leukocytes on the surface of plaques from PMio exposed rabbits. a) A leukocyte protruding from three openings in the endothelium, b) Inset from panel a. Small endothelial filopodia can be seen extending up the side of the endothelial cell (arrow), c) A migrating leukocyte, d) Inset from panel c. The leukocyte is emerging between two endothelial cells (endothelial cell 1 (EC1) and EC2) with the endothelial border extending down the side of the cell (black arrows). Small filopodia from EC1 extend up the side o f the leukocytes (white arrows), e) Surface of a migrating leukocyte, f) Inset of panel e. N o endothelial cell extensions visible on the side of the leukocyte. 124 Chapter 6: Surface Morphology o f Atherosclerotic Plaques Figure 6.15. Emigrating macrophage-derived foam cell, a) A large macrophage-derived foam cell erupting out from the blood vessel wall . Surrounding endothelial cells (EC) are pushed outwards into the lumen. Scale bar = 5 um. b) A n inset o f the luminal aspect of the macrophage-derived foam cell that is carrying some of the reticulum of dense E C M out into the lumen. The tip of an E C is left attached to the migrating cell (arrow). Scale bar = 2 pm. c) The emigrating cell has lifted the endothelium up, pushed it into the lumen and appears to be curling over the top o f the endothelial cell. Scale bar = 2 pm. d) The endothelium has been pushed up into the lumen and folded back on itself altering the topography o f the vessel. Scale bar = 2 pm. 125 Chapter 6: Surface Morphology of Atherosclerotic Plaques Figure 6.16. Cell immigration into an atherosclerotic lesion, a) A large cell with some lipid accumulation attached to the surface of an atherosclerotic lesion. Scale bar = 5 pm. b) Inset of a. The endothelial cells (EC) are visible, being pushed down into the atherosclerotic lesion as the cell transmigrates. Scale bar = 2 pm. c) Inset o f panel b. A long extension o f the migrating cell (arrows) separating two endothelial cells which are angled down into the atherosclerotic lesion. Scale bar = 2 pm. 126 Chapter 6: Surface Morphology o f Atherosclerotic Plaques S4700 2 5kV 11 9mm x7.00k SE(U) 8/5/05 5 OOum S4700 2 51V 11 9mm x40 Ok SE(U) 8/5/05 1 OOum Figure 6.17. Endothelial erosion and subsequent platelet adhesion on exposed extracellular matrix, a) A n area of endothelial desquamation, b) Lower inset of panel a. Platelets are adhering to the exposed underlying E C M . c) Upper inset o f panel a. Platelet adhesion and spreading on the exposed E C M . d) Detailed image o f the reticulum of dense E C M exposed during endothelial erosion. 6.5 Discussion S E M analysis of plaques from control and PMio exposed W H H L rabbits revealed patches of intense leukocyte adhesion and transmigration over plaque cores of the PMio exposed rabbits. As expected leukocyte adhesion and transmigration were also observed at the shoulder regions of atherosclerotic plaques from both control rabbits and PMio exposed W H H L rabbits. The size, and cytoplasmic extensions of the transmigrating cells suggests they are macrophage-derived foam cell. However, to our surprise, the morphology o f macrophage-derived foam cell / endothelial cell interactions, suggest that most were actually emerging from the central regions of the plaques of PMio exposed rabbits. The surface topography o f the endothelium over the core regions of atherosclerotic plaques was irregular with 127 Chapter 6: Surface Morphology o f Atherosclerotic Plaques intercellular gaps in the endothelium, appearing different than the regular intact endothelium observed from areas of normal blood vessel wall . The alterations in endothelial topography between diseased and non-diseased areas o f the arterial wall were expected. Atherosclerosis is a disease state, which in its advanced stages results in a narrowing of the blood vessel lumen. 1 4 6 With this narrowing comes changes in blood flow and altered hemodynamics both of which are important contributors to endothelial shape, arrangement and topography. 1 4 1 The S E M observation of atherosclerotic plaque endothelium elevated into the lumen of the blood vessel at a greater height than that of endothelial cell nuclei, and the T E M observation of large l ipid engorged foam cells pushing the endothelium into the lumen are believed to be the same process. The large mounds about 1 0 - 1 5 pm in size observed during S E M analysis are about the same size as subendothelial macrophage-derived foam cells observed during T E M analysis that produced a mounded appearance to the endothelium. Mounded endothelium has been previously described at atherosclerotic plaque shoulders by Nakamura and colleagues though they did not speculate as to the cause of the mounding. Our observation o f a population of adherent and transmigrating leukocytes is not novel in the atherosclerotic disease state. 2 9 5 However, what is unique is the differential adhesion to and transmigration through the core endothelium observed between the control rabbits and the P M i o exposed rabbits. Atherosclerosis is a disease in which plaque advancement typically occurs from the shoulder regions. A s such, the observation of active cell adhesion and transmigration at the shoulder regions in control and PMio exposed hyperlipidemic rabbits 'yon O Q C was expected. ' It was considered evidence of advancing plaques under the stimulus o f hypercholesterolemia in the W H H L rabbit model. Furthermore, it has been previously reported that PMio exposure resulted in more extensive atherosclerosis. 1 0 6 B y contrast, the core regions o f atherosclerotic plaques are typically more stable than the shoulder regions, characterized by a fibrous cap reinforced by E C M instead of active macrophage migration. The observation made herein of adherent and transmigrating leukocytes over the core regions of plaques in addition to the shoulder regions of atherosclerotic plaques in the PMio exposed W H H L rabbits is novel. Even more intriguing are the observations from both T E M and S E M 128 Chapter 6: Surface Morphology of Atherosclerotic Plaques analyses suggesting that the majority of migration on the core of the atherosclerotic plaques in the PMio exposed rabbits was actually emigration out of the plaques into the arterial lumen. In 1981, Gerrity and colleagues 2 8 9 used T E M to show large macrophage-derived foam cells distending the endothelium into the lumen. They hypothesized that the observation of the endothelium being pushed outwards was evidence of cell emigration and suggested that emigration may represent an attempt to clear accumulated l ipid from the atherosclerotic plaque, yet this observation did not include actual evidence of transmigration into the lumen. Our report o f actual leukocyte emigration from atherosclerotic plaques lends new credibility to their hypothesis. The observation suggests the cap regions o f atherosclerotic plaques from PMio exposed rabbits become an area of active remodelling apparently driven by macrophage-derived foam cells emigrating into the vascular lumen. It is tempting to suggest that the low grade chronic inflammatory response documented following PMio exposure may in fact provide the chemokine stimulus for the emigration of macrophage-derived foam cells out o f the atherosclerotic plaque and into the lumen of the blood vessel through chemokine penetration of plaque endothelium. Though many adherent and transmigrating cells were observed on the shoulders of atherosclerotic plaques, very few were small enough to be immigrating monocytes. It has been shown that the process of transmigration for a neutrophil takes a very short amount o f time, typically less than 2 minutes though it can be as fast as 30 seconds. Though the transmigration time of a monocyte is unknown, we assume it is of a similar duration. If this is in fact the case, it could explain why very few monocytes were observed in the process o f immigration; it is such a rapid process that the probability o f catching a cell transmigrating is low. B y contrast we would expect that the large macrophage-derived foam cells would take longer to emigrate such that the probability of observing emigrating macrophage-derived foam cells would be greater. Furthermore, emigrating macrophage-derived foam cells are several times larger than monocytes / macrophages and in order to emerge from the atherosclerotic plaque these cells must make their way through the various cellular and E C M components o f the fibrous caps. 129 Chapter 6: Surface Morphology of Atherosclerotic Plaques In the infrequently observed areas of endothelial denudation, platelets were adherent to the underlying E C M components. This observation suggests that the endothelial denudation seen was actually a biological event occurring prior to animal sacrifice and tissue processing rather than an artefact of those processes. This affirms the prothrombotic nature of the novel reticulum o f dense E C M as well as its l ikely basal lamina type constituents known to also be 7Q7 TOO pro-thrombotic. " Furthermore, the images taken of the E C M in areas of endothelial denudation confirmed the reticular organization of the dense E C M illustrated in the 3-dimensional serial reconstructions in Chapter 4. The observations in this chapter constitute a novel description of leukocyte emigration following PMio exposure rather than the expected monocyte immigration. In Chapter 3, the accumulation of macrophage-derived foam cells was observed in the subendothelial region of plaque cores. Then in Chapter 4, that accumulation was discovered to result in degradation o f the unique reticulum of dense E C M that permitted increased direct contact between macrophage-derived foam cells and the endothelial cells. In Chapter 5, histochemical staining confirmed the presence of proteoglycans likely to include basement membrane constituents where we ultrastructurally observed the reticulum o f dense E C M in atherosclerotic plaques from control W H H L rabbits and its apparent degradation in P M i o exposed rabbits. This unique scenario is discussed in detail in Chapter 7 where a model for the whole process is presented. In this chapter, evidence has been provided that over these core regions o f atherosclerotic plaques from PMio exposed rabbits macrophage-derived cells were actually emigrating not immigrating as expected. It appears as i f exposure to P M i o has led to destabilizing changes in atherosclerotic cap composition as the result o f the mobilization of the macrophage-derived foam cells from deep within the core regions o f atherosclerotic plaques into the systemic circulation. 130 CHAPTER 7 Summary and Discussion 7.1 Restatement of the Problem A i r pollution related mortality is responsible for in excess of 2,000,000 deaths annually, of which acute cardiovascular events account for the greatest proportion. 1 The primary cause o f all acute cardiovascular events is rupture o f unstable atherosclerotic plaques and subsequent thrombus formation. 1 6 5 " 1 6 7 ' 3 0 0 Features deemed to be characteristic o f an unstable atherosclerotic plaque include increased deposition of l ipid, a large population of macrophage-derived foam cells, a small population of S M C s and a reduction in E C M . 1 6 5 ' 3 0 0 One of the stimuli to convert stable atherosclerotic plaques into unstable atherosclerotic plaques includes a chronic inflammatory response, a response reported in both animal and human investigations following exposure to P M i o . 9 5 ' " ' 1 0 4 ' 1 0 5 The unresolved question in relation to PMio attributed morbidity and mortality is: which of the features o f an unstable atherosclerotic plaque outlined above are observed in atherosclerotic plaques following exposure to PMio? To date, only two investigations have been published addressing that question. The first was a 2002 paper by Suwa and colleagues. 1 0 6 They observed a trend toward larger atherosclerotic plaques, and greater numbers of atherosclerotic plaques with type III, IV and V plaques pathological classifications. The suggestion of advancing atherosclerotic plaques was founded on observations of increased infiltration of inflammatory cells as well as increased levels of l ipid accumulation in atherosclerotic plaques from W H H L rabbits exposed to PMio . In agreement with the Suwa observations, 1 0 6 a 2004 paper by Sun 101 and colleagues reported increased amounts of l ipid and an increased population o f CD68 positive macrophages in the intima and media o f plaques from A p o E -/- mice fed high fat chow and exposed to low levels o f PM2.5. B y demonstrating increased plaque lipids and inflammatory cell invasion, these two preliminary studies established evidence o f changes in atherosclerotic plaques following PMio exposure that were consistent with the more 131 Chapter 7: Summary and Discussion vulnerable atherosclerotic plaque phenotype. However, the accumulation of inflammatory cells and increased lipid content are only two o f a number of phenomena associated with increasingly vulnerable atherosclerotic p laques . 1 6 2 ' 1 6 9 The purpose of this study was to use electron microscopy to examine atherosclerotic plaques from W H H L rabbits exposed to PMio in order to resolve the features o f the remodelling and reorganization process. The main emphasis o f this work was placed on the integrity of the endothelium and its underlying E C M , the redistribution of cell populations within the atherosclerotic plaque, evidence o f cap remodelling, and leukocyte transmigration, all features not addressed in two previous studies. 106, 107 7.2 Summary of Findings This dissertation details alterations in specific aspects of atherosclerotic plaque structure and composition that directly contribute to decreasing plaque stability. In atherosclerotic plaques from PMio exposed W H H L rabbits, we demonstrated 1. an accumulation of macrophage-derived foam cells immediately below the endothelium of atherosclerotic plaque cores; 2. ultrastructural evidence of the separation of the endothelium from the heretofore undescribed unique reticulum of dense E C M that serves as the supporting basal lamina of atherosclerotic plaques; and, 3. evidence of degradation or fragmentation of the reticulum o f dense E C M in regions o f macrophage-derived foam cell accumulation. A s a consequence o f fragmentation o f the E C M there was increased direct contact between macrophage-derived foam cells and the abluminal plasma membrane of endothelial cells. A l l of these 2-dimensional observations were confirmed through 3-dimensional serial section reconstructions and S E M observations. Histochemical techniques established the presence of a layer of acidic and sulphated proteoglycan moieties along the atherosclerotic plaque surface confirmed by T E M to be the reticulum of dense E C M . In PMio exposed rabbits, the layer of acidic and sulphated proteoglycan moieties appeared discontinuous or absent along the atherosclerotic plaque surface. Further immunohistochemical investigations are required to determine whether this layer o f proteoglycan moieties contains laminin, type IV collagen and basal lamina specific proteoglycans. While histochemical techniques were used to address compositional changes in the reticulum of dense E C M under the endothelium, S E M and T E M were used to investigate cell transmigration across the atherosclerotic cap endothelium. Increased 132 Chapter 7: Summary and Discussion macrophage-derived foam cell transmigration was observed over the core regions of atherosclerotic plaques from PMio exposed rabbits and the evidence suggests the cells were emigrating. Careful consideration of the observations made above has led us to propose that the accumulating macrophage-derived foam cells, degradation of the E C M and increased macrophage-derived foam cells traffic across the endothelium of atherosclerotic plaques following exposure to PMio are stages of a process of mobilization of the macrophage-derived foam cell population from the core regions of atherosclerotic plaque to the atherosclerotic plaque cap region and then into the systemic circulation. Emigrating macrophage-derived foam cells and degradation of the E C M constitute new evidence o f atherosclerotic plaque destabilization as a consequence of PMio exposure. 7.3 Findings in the Context of the Scientific Literature 7.3.1 Macrophage-Derived Foam Cell Accumulation Our first novel finding was an accumulation of macrophage-derived foam cells immediately below the endothelium o f atherosclerotic plaque cores in PMio exposed rabbits. It is known that PMio exposure is accompanied by a chronic inflammatory response that includes increased numbers of circulating leukocytes and cytokines. 1 0 4 The generally held view o f atherosclerotic plaque growth and development stipulates that IL-6, T N F - a , and G M - C S F stimulate circulating leukocytes, particularly monocytes, resulting in a proatherogenic state o f the blood and increased monocyte infiltration into the blood vessel in t ima. 1 1 8 ' 1 3 7 The phenomenon o f monocyte adhesion and immigration into atherosclerotic plaques 2 8 7 where they internalize extracellular oxidized L D L 2 6 1 to become macrophage-derived foam cells is believed to occur principally at the shoulder of enlarging plaques. 2 8 7 Macrophage-derived foam cells are known to produce T N F - a , I L - l a , IL-1 p, IL-6, IL-8, IL-10, IL-12, IL-15, IL-18, G M - C S F , M - C S F , M C P - 1 and TGF-p , as well as a spectrum of M M P s including M M P - 1 , -2, -3, -7, -8, -9 and - 1 3 . 1 3 7 ' 1 4 8 ' 1 5 4 > 3 0 1 ' 3 0 2 A l l o f these cytokines and enzymes are involved in atherosclerotic plaque progression and destabilization, via E C M breakdown, stimulation o f the bone marrow, activation of monocyte migration, endothelial cell activation and S M C activation and migration. 1 3 7 M C P - 1 is a known chemoattractant capable of stimulating 133 Chapter 7: Summary and Discussion macrophage-derived foam cell chemotaxis. Our observation in PMio exposed rabbits of an accumulation of macrophage-derived foam cells immediately below the endothelium of atherosclerotic plaque cores constitutes a new finding relevant to the identification of possible mechanisms of PMio induced atherosclerotic plaque vulnerability. Sites of atherosclerotic plaque rupture have been associated with monocyte accumulation and M M P p r o d u c t i o n . 1 5 4 ' 3 0 4 ' 3 0 5 It is tempting to suggest that a driving factor behind this phenotype of a subendothelial accumulation of macrophage-derived foam cells could be the permeation of the already compromised endothelium by macrophage-derived foam cell chemoattractants. 7.3.2 Separation of the Endothelial Cells from the E C M and Its Degradation Given the known activities of macrophage-derived foam cells, the previously unreported changes in plaques of PMio exposed W H H L rabbits that we observed around the accumulating macrophage-derived foam cells make sense. The most interesting and novel observations we made in plaques of PMio rabbits were the: 1. separation o f endothelium from its unique reticulum of dense E C M ; 2. fragmentation of that reticulum of dense E C M ; and 3. increased direct contact between macrophage-derived foam cells and the abluminal plasma membrane of endothelial cells. Normal arterial endothelium and its multiple functions, including immunologic, secretory, synthetic and metabolic functions, 2 3 1 may be compromised as a consequence of the atherosclerotic cap reorganization and the leukocyte emigration reported here. A primary 230 231 function of the endothelium is to regulate both cellular and solute trafficking. ' The endothelium of an unaffected artery is usually supported by its subtending basal lamina, which is composed of type IV collagen, laminin and proteoglycan constituents that provide critical mechanical support, a prerequisite for endothelial fulfilment of the above 998 990 9^9 9^8 functions. ' ' " Normal hemodynamic stresses result in the expression of atheroprotective genes necessary for the maintenance of a healthy vessel w a l l . 1 4 2 Under high shear stress, endothelial cells appear more elongated, microfilaments and intermediate filaments are increased, and E C M th in s . 1 4 1 ' 1 4 3 With reduced attachment to the extracellular matrix o f the basal lamina, the subtending endothelium becomes more vulnerable to 134 Chapter 7: Summary and Discussion In atherosclerotic plaques from control W H H L rabbits we observed a unique 3-dimensional organization of a reticulum of dense E C M subtending the endothelium in place of the basal lamina. This reticulum of dense E C M appears in one ultrastructural study o f aortic atherosclerotic plaques, 2 5 5 but it was not discussed. The closest thing to the observed reticulum o f dense E C M in plaques is found in the vasculature of diabetics. 2 7 0 Furthermore, the nature, composition, and origin of the thickened reticulum of dense E C M has not been characterized. In atherosclerotic plaques from PMio exposed W H H L rabbits, we observed the separation of the endothelial cells from the reticulum of dense E C M , as well as fragmentation and mechanical disruption of the dense reticulum of E C M in association with an accumulation o f macrophage-derived foam cells. A s a consequence of the fragmentation or absence of E C M , the abluminal plasma membrane of the endothelial cells was more frequently put in direct contact with the macrophage-derived foam cells. S E M and T E M observations supported the suggestion of macrophage-derived foam cell emigration at plaque surfaces; as such, the major contact substrate for the endothelial cells was a layer of apparently migrating macrophage-derived foam cells. Stable attachment surfaces for the endothelium were either absent or significantly reduced following PMio exposure. Separation of the endothelium from the reticulum of dense E C M , degradation of the reticulum, and increased direct contact between macrophage-derived foam cells and endothelial cells are all characteristics consistent with increased atherosclerotic plaque vulnerability. Such changes in atherosclerotic plaque organization have not been previously reported for plaques of P M i o exposed rabbits. Although a rupture or Assuring event is two to three times more l ikely to cause a major thrombus than plaque e r o s i o n , 1 6 2 ' 3 0 6 any alteration in the stability of the endothelium and its subtending E C M as seen here could still contribute to acute events such as a myocardial or cerebral infarction, both events associated with PMio exposure. 7.3.3 Reduction of Acidic and Sulphated Proteoglycan Moieties Although the reticulum o f dense E C M subtending the plaque endothelium is ultrastructurally similar to a thickened basal lamina, its unique organization suggests the possibility that it could be made of different constituents or contain additional elements. Therefore, we began 135 Chapter 7: Summary and Discussion to define its histochemical makeup. Staining for neutral, acidic and sulphated carbohydrates moieties established the presence of a layer of acidic and sulphated proteoglycan moieties along the atherosclerotic plaque surfaces of the control rabbits. This material lies immediately below where the endothelium sits on the plaque surfaces. Presumably, because o f damage due to handling, the endothelium was removed. This view was supported by ultrastructural inspection of these plaques that revealed the outermost layer to be a reticulum of dense E C M as we described in Chapter 4. This layer appeared discontinuous or absent from plaque surfaces and from between cells within the cap region of PMio exposed rabbits. These histochemical findings are in agreement with our ultrastructural description of the reticulum of dense E C M and its apparent degradation in Chapter 4. Microanatomical staining 979 allows the identification of most major proteoglycans including perlecan and basement membranes materials such as laminin, collagen, and reticular fibers. 2 7 1 The loss o f these intercellular moieties in the fibrous cap region of PMio exposed rabbits fulfills another criterion of atherosclerotic plaque destabilization; namely reduced E C M c o n t e n t . 1 6 2 ' 2 1 4 ' 2 4 8 ' 2 4 9 These observations are significantly more refined than any of the previous studies including those from our own laboratory. 7.3.4 Macrophage-Derived Foam Cell Emigration Perhaps the most intriguing finding in this study was the observation of increased foam cell transmigration over core regions of atherosclerotic plaques from PMio exposed rabbits. Monocyte immigration at the shoulder regions of atherosclerotic plaques is an established aspect of plaque progression. 2 8 7 That macrophage-derived foam cells are actually emigrating from atherosclerotic plaques of PMio exposed rabbits is a novel observation that has only been suggested once before in the literature and for different stimuli other than P M i o exposure. 2 8 9 Unexpectedly, all leukocytes observed by S E M and T E M adherent to or transmigrating through plaque endothelium were macrophage-derived foam cells. Three observations support the identification of these leukocytes as macrophage-derived foam cells: 1. extensive cytoplasmic extensions on the surface of these cells. 2. their large size, and 3. in T E M sections of migrating cells, large accumulations of l ipid droplets are present. In contrast to evidence in the literature, we did not observe monocyte immigration, but rather macrophage-derived foam cell emigration from plaque surfaces. Close examination of the 136 Chapter 7: Summary and Discussion aperture edges through which these macrophage-derived foam cells were transmigrating revealed numerous thin endothelial filopodia extending up to apparent focal adhesion contacts on the surface of such foam cells. T E M revealed that macrophage-derived foam cells protruding through the endothelium into the lumen carried along small masses of the reticulum o f dense E C M . These observations are all in accord with the proposition that macrophage-derived foam cells were emigrating from the surfaces of PMio exposed atherosclerotic plaques. The novel observation that leukocyte transmigration at the surface of atherosclerotic plaques is actually leukocyte emigration raises a number of important questions. For instance, where do these macrophage-derived foam cells that accumulate below the plaque endothelium come from? What are the consequences of macrophage-derived foam cell migration through the atherosclerotic plaques in so far as the integrity of fibrous caps and subendothelial E C M ? What are the consequences of macrophage-derived foam cell emigration across the endothelium with regards to endothelial integrity? Where do the emigrating foam cells go, and with what consequences? 7.3.5 Relevance of the W H H L Rabbit Model Are our observations of altered endothelial integrity due to subendothelial macrophage-derived foam cell accumulation and emigration from atherosclerotic plaques in a rabbit relevant to human exposure to PMio? The W H H L rabbit is an endogenous model of hyperlipidemia resulting from a genetic defect in the L D L receptor gene. 2 0 3 ' 2 0 4 The pathophysiology of this genetic defect in the rabbit is very similar to the human condition o f familial hypercholesterolemia 2 0 4 which is an autosomal dominant, inherited deficiency o f the L D L receptor resulting from many known mutations. 3 0 7 In human populations, homozygotic familial hypercholesterolemia has a prevalence of 1/1,000,000 whereas heterozygotic familial hypercholesterolemia has a prevalence of 1/500. 3 0 7 On a normal chow diet, the homozygotic W H H L rabbits develop atherosclerotic plaques that are indistinguishable from human atherosclerotic plaques exhibiting features such as fibrous caps, l ipid cores and inflamed intimal involvement. 2 0 5 In the heterozygotic W H H L rabbit model, cholesterol feeding is required, but these animals also develop atherosclerotic plaques that resemble 137 Chapter 7: Summary and Discussion those found in humans. High cholesterol diets in most human populations and the more common heterozygotic genotype may have more similarity to the heterozygotic W H H L rabbit model than the homozygotic W H H L rabbit model. The model used for the series of studies described in this dissertation is the homozygotic W H H L rabbit. One challenge associated with the W H H L rabbit is the well documented inconsistency o f plaque formation. O f the regions of the aorta, the thoracic aorta is more prone to atherosclerotic plaque formation than the abdominal aorta. 2 0 4 However, sampling for this study was confined to the abdominal aorta to maximize tissue sample availability and to maintain statistical power under multi-user conditions. Our findings of variable plaque formation in the abdominal aortas of W H H L rabbits is not surprising given previous observations in the literature. 2 0 4 Future studies should focus on the thoracic aorta to maximize the availability of atherosclerotic plaques for investigation and to extend our observations to the more diseased aortic segment. Another issue regarding the relevance of the W H H L atherosclerotic rabbit model to human disease atherosclerosis is that human atherosclerotic plaque rupture may occur far more frequently in the coronary circulation than in the aorta. However, plaque rupture is not limited to the coronary circulation. In this dissertation, we used tissues from the abdominal aorta in which to study morphological alterations of atherosclerotic plaques following P M i o exposure. Our reasoning for this is as follows: to begin, fatty streak development is not initiated in the coronary vasculature until a minimum of 5 months of age in the W H H L rabbit 707 model, and it is not until 2-3 years of age that more than 50% of the W H H L rabbits have advanced coronary artery disease, typically located at the proximal origin of the left coronary artery. 3 0 9 In order to study alterations in atherosclerotic plaque organization, advanced plaques were needed in both the control and the PMio exposed rabbits. The long time-course required for advanced lesion development and the small surface area of the coronary circulation was not suitable for the goals of this study. The aortic arch is known to have the most rapid atherosclerotic plaque development in the W H H L rabbit model, followed closely by plaque development in the thoracic aorta. 2 0 7 However, due to efforts to maximize tissue use, in most cases the aortic arch and thoracic aorta were used for other studies. 138 Chapter 7: Summary and Discussion Atherosclerotic plaque development in the abdominal aorta occurs more rapidly than in the coronary circulation, yet more slowly than in the aortic arch and thoracic aorta. Although the prevalence of atherosclerotic disease in the abdominal aorta of the WHHL rabbits was lower than in the thoracic aorta, the greatest number of WHHL rabbit tissue samples originated from the abdominal aorta. Because the chronic inflammatory response that results following PMio exposure is systemic and not limited to one specific region of the vasculature, we have assumed that observed changes in the abdominal aortic plaques would eventually occur within the coronary vasculature. Further, if the atherosclerotic plaques in the abdominal aorta tend to develop more slowly than in other regions of the vasculature, it stands to reason that observed changes in the abdominal aorta may be more pronounced in the thoracic aorta and aortic arch. Ideally, future studies would include sampling of other areas of arterial circulation to test these assumptions. All of the observations for this study arise from WHHL rabbits that were sacrificed after exposure twice a week for four weeks. Cardiovascular events resulting from plaque rupture and thrombosis formation due to myocardial or cerebral infarction may occur within hours of a spike in PMio exposure. Superficially, it would appear that our WHHL rabbit model is a chronic exposure model, while the cardiovascular events in humans are a result of acute exposure. There is evidence to suggest that humans affected by PMio exposure already had pre-existing atherosclerotic disease. " Another point to be made is that suddenly increased levels in air pollution and PMio exposure in the urban environment are not likely to occur on a background of no prior exposure to PMio. Data on PMio exposure clearly indicates that any exposure is potentially deleterious and could foster low grade systemic inflammation. Ultimately, the method to address this limitation would be a time-course study that included sacrificing WHHL rabbits immediately following their first exposure, and within days and following months of repeated exposure. The expense of animals and their inclusion in a pre-existing principal investigation precluded doing such a time-course for the benefit of this study. While a time-course would improve our ability to interpret the data, our findings of significant compromises in plaque stability with this model of PMio exposure are still of great significance and, if nothing else, argue for the necessity of further explorations of this phenomenon. 1 3 9 Chapter 7: Summary and Discussion 7.4 Proposed Mechanisms for PMi0 Induced Atherosclerotic Plaque Destabilization When the findings presented in this dissertation are considered in the context of previous data as to the effects of PMio on atherosclerotic plaque vulnerability, it is now possible to outline the potential mechanisms by which exposure to PMio leads to atherosclerotic plaque destabilization and ultimately plaque rupture. This novel scenario also suggests an explanation for some of the observed changes in plaque organization following P M i o exposure. Stable atherosclerotic plaques are characterized by a thick fibrous cap composed of an overlying layer of endothelial cells, their subtending reticulum of dense E C M and a matrix o f S M C s and E C M overlying a necrotic core (refer to Chapter 3 for further details). Typical ly this core contains macrophage-derived foam cells and gruel composed of extracellular l ipid, cell debris and occasional S M C s (Figure 7.1a). In response to PMio deposition in pulmonary alveoli, alveolar macrophages phagocytize the instilled particles, become activated in the process and release cytokines such as IL-6, T N F - a and G M - C S F into the a l v e o l i . 1 0 4 , 1 7 5 The cytokines cross into the systemic circulation and have been shown to stimulate the bone marrow to release band cells (immature neutrophils) and P M N into the systemic circulation. 1 0 6 , 1 7 8 These circulating cytokines and increased circulating leukocytes constitute the components of a systemic inflammatory response. 2 2 2 In addition to activating the bone marrow, the cytokines circulating in the plasma may be able to either penetrate the typically compromised plaque endothelial cell barrier or stimulate these endothelial cells to release cytokines chemotactic to macrophage-derived foam cells. In response to these cytokines infiltrating into the atherosclerotic plaque the macrophage-derived foam cell population may thus be stimulated to migrate towards the endothelium and ultimately into the blood vessel lumen (Figure 7.1b). A s the macrophage-derived foam cells are stimulated to migrate, they would l ikely secrete M M P s that would be capable of degrading the E C M components of the atherosclerotic 140 Chapter 7: S u m m a r y and D i s c u s s i o n plaque i n c l u d i n g the r e t i c u l u m o f dense E C M , a l l o w i n g their easy passage towards the e n d o t h e l i u m . 3 0 5 ' 3 1 0 T h i s m i g r a t i o n o f macrophage-derived f o a m cells w o u l d result i n an increase i n macrophage-derived f o a m cells b e l o w the endothel ium (Chapter 3) and a decrease Figure 7.1 Altered plaque organization following P M i o exposure, a) Characterist ics o f a stable atherosclerotic plaque inc lude a th ick f ibrous cap c o m p o s e d o f an outer layer o f endothel ia l cel ls , a re t icu lum o f dense E C M and a matr ix o f S M C s a n d E C M o v e r l y i n g a large necrotic core conta in ing macrophage-der ived f o a m cel ls and gruel . T h e internal elastic l a m i n a ( I E L ) separates the plaque core f r o m the S M C r i c h m e d i a reg ion o f the b l o o d vessel w a l l , b) F o l l o w i n g P M i o exposure, there is a decrease i n the p o p u l a t i o n o f macrophage-der ived f o a m cel ls w i t h i n the atherosclerotic core l e a v i n g a necrot ic , acel lular gruel behind, a n d an increased populat ion o f macrophage-derived f o a m cells subtending the endothel ium, and emigrat ing f r o m the plaque into the vascular l u m e n . 141 Chapter 7: Summary and Discussion in the population of macrophage-derived foam cells within the atherosclerotic core, leaving a necrotic, acellular gruel behind (supported by qualitative observations from Chapter 3). The infiltration and accumulation of the macrophage-derived foam cells would lead to a decrease in the apparent cap density of S M C s due to the dilution of the S M C population among a growing population of macrophage-derived foam cells (Chapter 3) coupled with the M M P degradation of the surrounding E C M (Chapter 5). A long with this overall cellularity of the fibrous cap, the l ipid content increases. This redistribution of l ipid by the migrating cells may make it appear as i f the l ipid content of the entire atherosclerotic plaque has increased, given that the extracellular lipids of the core would have been left behind. In addition, the accumulating macrophage-derived foam cells immediately below the endothelium separate the endothelial cells from the reticulum of dense E C M while they degrade it by emigration activities and mechanical disruption (Chapter 4). The M M P enzymatic activities would also open up space in the E C M of the fibrous cap surrounding the S M C (Chapter 5). This accumulation o f macrophage-derived foam cells and the separation of the endothelium from its supporting E C M would distend the endothelium into the vessel lumen. Furthermore, this subendothelial macrophage-derived foam cell presence would alter plaque surface topography as would macrophage-derived foam cell transmigration through the plaque endothelium (Chapter 6). The macrophage-derived foam cell transmigration might also alter endothelial barrier functions and superficial hemodynamics on the plaque surface. 7.5 Proposed Mechanism of PMi0 Induced Morbidity and Mortality In the introduction, three potential mechanisms were introduced by which particulate matter air pollution may bring about acute cardiovascular events. Those were the inflammatory, the dysfunction of the autonomic nervous system and the cardiac malfunction mechanisms. 5 4 I would propose the idea that the underlying cause is not one of the above mechanisms, but rather the result of the interactions of all three, particularly the inflammatory and autonomic dysfunction mechanisms. The reasoning behind this proposed combination o f factors is as follows: first the studies described in this' dissertation have identified structural changes in the atherosclerotic cap following PMio exposure. The cause o f the alterations appears to be the stimulated migration 142 Chapter 7: Summary and Discussion of macrophage-derived foam cells out of the atherosclerotic cores, into the cap region, and eventually into the blood vessel lumen. As a consequence of the migrating cells and their production of MMPs, the structural ECM of the atherosclerotic cap is degraded and the protective stratification of the plaque is lost. The result is vulnerable atherosclerotic plaques that are susceptible to rupture. If you will, imagine an individual who is aging, who has at least one or two major risk factors for atherosclerosis and who has been chronically exposed to low levels of air pollution. Epidemiological investigations repeatedly link PMio exposure to adverse cardiovascular events in individuals with a prior myocardial infarction88' 1 0 9 ' I 1 0 ' 3 1 1 or chronic insulin-resistant diabetes,111 both of which are associated with advanced atherosclerotic disease. Evidence presented throughout this dissertation suggests that the atherosclerotic plaques in an individual undergo destabilizing structural changes as a consequence of exposure to PMio. Combined, the basic science and the epidemiological studies suggest that an individual will experience the progressive worsening and destabilization of their atherosclerotic plaques as a consequence of PMio exposure. Now imagine there is a thermal inversion, or a neighbouring forest fire, or something unexpected that ultimately results in an increase in airborne PMio levels. Epidemiology and basic science investigations suggest that at this point there will be a decrease in heart rate variability94 and an increase in circulating white cells and proinflammatory cytokines,95'106 heart rate,96 cardiac arrhythmias,93 systolic blood pressure,97 arterial vasoconstriction, plasma viscosity, platelet activation, and fibrinogen levels. Combined, all of these factors result in a procoagulant and proinflammatory state of the circulating blood. What we have now in our hypothetical individual is vulnerable atherosclerotic plaques, increased reactivity of the blood and altered shear stress on the vasculature, a perfect deadly scenario for an acute cardiovascular event to play out as follows: as the blood pressure increases, the flow properties of the blood may change, and a vulnerable atherosclerotic plaque, whose endothelium has lost its ECM substrate and had its topography altered by emigrating foam cells, may rupture or fissure. The increased blood viscosity, fibrinogen levels and platelet activation aids in large thrombus formation. If the thrombus does not fully occlude the arterial lumen, it may break off in the setting of increased luminal narrowing and vascular tone and be sent downstream in the arterial tree. 143 Chapter 7: Summary and Discussion More l ikely perhaps an occlusive thrombus may lead to a myocardial or cerebral infarction, and ultimately the death of the patient. Our hypothetical person has now become one of the 2 mil l ion people to die annually from air pollution exposure. The mechanisms involved in the death of our hypothetical person were not only the inflammatory mechanisms, but also the autonomic dysfunction mechanism. These two mechanisms are not separate; rather they are convergent processes that together lead to plaque destabilization and rupture. 7.6 Concluding Remarks A n accumulation of macrophage-derived foam cells subtending the endothelium, decreased endothelial contact with a dense, stable reticulum of E C M material and the emigration of large leukocytes from atherosclerotic plaques are indicators of atherosclerotic plaque remodelling following PMio exposure. Taken together with previous work, these findings demonstrated mechanisms that may lead to the remodelling o f stable atherosclerotic plaques to the vulnerable unstable atherosclerotic plaque phenotype following exposure to PMio . Cardiovascular disease has an enormous global disease burden and it appears that PMio exposure may be a considerable contributor to endothelial destabilization and dysfunction potentially resulting in millions of deaths annually. Although PMio exposure is not usually considered an initiating risk factor for atherosclerosis, it definitely appears to be an exacerbating risk factor, pushing existing atherosclerotic plaques to a vulnerable phenotype. A s with many risk factors for atherosclerosis, there are steps that can be taken to minimize the impact of PMio exposure and they would include steps to prevent atherosclerotic plaque formation along with measures to reduce levels of PMio in our atmosphere. A recent study reported 65% of young adults could not name a single risk factor for atherosclerosis. 3 1 2 Recent surveys report record numbers of Canadians are concerned about climate change and air pollution, yet only 2.2% are wi l l ing to consider the environment when they purchase a 313 new car. Increased education of the public and implementation of public policy needs to foster changes in population behaviours in order to reduce the morbidity and mortality rates associated with PMio exposure. ' 'It makes little sense to expect individuals to behave differently from their peers; it is more appropriate to seek a general change in behavioural norms and in the circumstances which facilitate their adoption'' -Geoffrey Rose 144 Chapter 7: Summary and Discussion 7.7 Future Directions A number o f arenas for further investigation and future studies of the effect of PMio exposure on atherosclerotic plaques in W H H L rabbits and other animal models become obvious: 1. Perform a time-course study of P M i o exposure in naive W H H L rabbits with robust atherosclerosis that follows the response of macrophage-derived foam cells at times that are used to study acute inflammation, e.g. 30 minutes, 1, 2, 4, 8, 12, 18, 24 hrs. A s well as, perform studies for longer periods of time, up to a year in duration to study the chronic effects of PMio exposure. 2. Examine human atherosclerotic plaques to determine the nature o f the E C M supporting the endothelium and how it compares to the reticulum o f dense E C M observed in the W H H L rabbits. 3. Repeat the experimental protocols described in this dissertation on existing thoracic aorta samples from W H H L rabbits exposed to PMio to confirm our observations. 4. Perform immunohistochemistry using antibodies designed for the W H H L rabbit model targeting type I V collagen, laminin, perlecan and versican on existing abdominal aorta and thoracic aorta tissue samples. 5. 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Surface area or Surface area Mass Volume The aerodynamic diameter of a particle is defined as the diameter of a spherical particle with a density o f l g / c m 3 (the density of water) that has the same settling velocity as the particle under consideration. 1 3 ' 1 4 A transition metal is any of the metallic elements that have an incomplete inner electron shell and that serve as transitional links between the most and the least electropositive in a series o f elements. They are characterized by multiple valences, coloured compounds, and the ability to form stable complex ions. 165 

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