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Matrix metalloproteinases, inflammation, and matrix remodeling in coxsackievirus-induced myocarditis Cheung, Caroline Tsui Yee 2007

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MATRIX METALLOPROTEINASES, INFLAMMATION, AND MATRIX REMODELING IN COXSACKIEVIRUS-INDUCED MYOCARDITIS by CAROLINE TSUI YEE CHEUNG B.Sc, The University of British Columbia, 2000 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA April 2007 © Caroline Tsui Yee Cheung, 2007 A B S T R A C T Myocarditis, or inflammation and injury of the heart muscle, induced by infection with coxsackievirus B3 ( C V B 3 ) is believed to lead to longterm heart disorders. Under pathological conditions, dysregulation of cardiac extracellular matrix ( E C M ) turnover results in maladaptive remodeling, progression o f disease, and depressed cardiac function. The mechanisms by which this E C M alteration occurs are unclear, but inflammatory cells may be involved as secretors of matrix metalloproteinases (MMPs) , cytokines, and growth factors that regulate E C M homeostasis. I hypothesize that M M P s play important roles in viral myocarditis. These enzymes degrade interstitial molecules, but they also regulate the immune system via modulation o f cytokines and growth factors. To investigate the proposed hypothesis, I first examined the expression profile o f M M P - 2 , M M P - 8 , M M P - 9 , M M P - 1 2 , and the tissue inhibitor of metalloproteinases (TIMPs), at key milestones in the evolution of myocarditis using a mouse model. To further explore the mechanisms, I infected M M P - 8 , M M P - 9 , and M M P - 1 2 knockout mice, and examined how these deficiencies affected the immune response and matrix remodeling. Lastly, I inhibited the 4 - I B B pathway, a major T-lymphocyte co-stimulatory molecule, to examine the synergy between the immune response and matrix remodeling. Following C V B 3 infection, increased M M P - 2 , M M P - 9 , and M M P - 1 2 expression was detected with corresponding decreases in T I M P expression, suggesting that an imbalance in M M P s and their inhibitors may result in increased protease activity. To examine the roles of the individual M M P s during C V B 3 infection, I infected mice lacking M M P - 8 , M M P - 9 , or M M P - 1 2 . Following viral infection, mice deficient in M M P - 1 2 and M M P - 9 experienced ii increased viral titers and tissue injury while MMP-8 knockout mice showed minimal differences as compared to controls. The mechanisms by which the M M P s operate may be in E C M remodeling, but also modulation of the immune response, notably T-cells. In order to determine the effect of the immune response on matrix remodeling, I inhibited the 4-IBB receptor, a major T-cell stimulatory pathway, and observed a decrease in inflammation and matrix remodeling, while M M P -12 was decreased, in association with an improvement in cardiac function. M y results suggest that M M P s may play crucial roles in C V B 3 infection through their immune modulation and matrix remodeling activities. iii TABLE OF CONTENTS A B S T R A C T i i T A B L E O F C O N T E N T S iv L I S T O F T A B L E S x L I S T O F F I G U R E S x i A B B R E V I A T I O N S x iv A C K N O W L E D G E M E N T S xv i i i C O - A U T H O R S H I P S T A T E M E N T xx C H A P T E R I: I N T R O D U C T I O N T O V I R A L M Y O C A R D I T I S 1 1.1 Overview o f Myocarditis 1 1.2 Definition of Myocarditis 2 1.3 Etiology o f Myocarditis 4 1.4 Diagnosis 11 1.5 Pathogenesis 14 1.5.1 Direct Virus-Mediated Injury 16 1.5.2 Immune Response 18 1.5.2.1 Innate Immunity 18 1.5.2.2 Adaptive Immunity 24 1.6 Reparation and Reclamation 33 1.6.1 Injury and Wound Healing in the Heart 33 1.6.2 Proteases and Matrix Remodeling 39 1.6.2.1 Cathepsins 40 1.6.2.2 Plasminogen System 42 iv 1.6.2.3 Matrix Metalloproteinases ( M M P s ) 45 1.7 Sequelae 51 1.8 Treatment 52 1.8.1 Immunosuppression 54 1.8.2 High-Dose Intravenous Immunoglobulin 56 1.8.3 Ant i -Vira l 58 1.9 Rationale, Hypothesis, and Experimental Aims 62 1.9.1 Rationale 62 1.9.2 Hypothesis 63 1.9.3 Specific Aims 63 1.9.4 Methodology Overview 63 1.9.5 Potential Significance o f Findings 65 1.10 References 66 C H A P T E R II: E X P R E S S I O N P R O F I L E O F M M P s A N D TIMPs I N C O X S A C K I E V I R U S - I N D U C E D M Y O C A R D I T I S 87 2.1 Rationale 87 2.2 Materials and Methods 89 2.2.1 Virus Preparation and Plaque Assay 89 2.2.2 Virus Infection of Animals 90 2.2.3 Quantitation of Vi ra l Titer 90 2.2.4 Histological Assessment 90 2.2.5 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) 91 2.2.6 Immunohistochemistry 92 2.2.7 Immunofluorescent Co-Localization 93 2.2.8 Gelatin Zymography 93 2.2.9 Western Blot 94 2.2.10 Data and Statistical Analyses 95 2.3 Results 95 2.3.1 Virus Detection and Histological Examination 95 2.3.2 Transcriptional Regulation of M M P s 97 2.3.3 Protein Expression and Localization of M M P s 101 2.3.4 Gelatinolytic Activation of M M P - 2 and M M P - 9 107 2.3.5 Protein Expression of TIMPs 107 2.4 Conclusions and Discussion 110 2.5 References 116 C H A P T E R III: A B L A T I O N O F M M P - 1 2 I N C V B 3 - I N F E C T E D M I C E I N C R E A S E S S E V E R I T Y OF M Y O C A R D I T I S , H E P A T I T I S , & P A N C R E A T I S 119 3.1 Rationale 119 3.2 Materials and Methods 120 3.2.1 In Vitro Study 120 3.2.1.1 Cel l Line 120 3.2.1.2 Virus Infection and Detection 121 3.2.1.3 Nucleic A c i d Extraction and P C R 121 3.2.1.4 Protein Extraction and Western Blot 122 3.2.2 In Vivo Study 122 3.2.2.1 Animals and Virus Infection 122 vi 3.2.2.2 Histological Assessment 122 3.2.2.3 Virus Detection and Plaque Assay 123 3.2.2.4 Irnmunohistochemistry 123 3.2.2.5 T U N E L Staining 123 3.2.2.6 Data and Statistical Analyses 124 3.3 Results 124 3.3.1 In Vitro Study 124 3.3.1.1 Cultured Murine Cardiomyocytes 124 3.3.1.2 Transcriptional and Translational Expression of M M P - 1 2 in HL-1 126 3.3.2 In Vivo Study 128 3.3.2.1 Morbidity and Histological Examination 128 3.3.2.2 Matrix Composition and Fibrosis 131 3.3.2.3 Virus Detection and Quantitation 135 3.3.2.4 Apoptosis .137 3.4 Conclusions and Discussion 138 3.5 References 146 C H A P T E R IV: M M P - 9 D E F I C I E N C Y I N C R E A S E S S E V E R I T Y O F V I R A L M Y O C A R D I T I S F O L L O W I N G C V B 3 I N F E C T I O N 149 4.1 Rationale 149 4.2 Materials and Methods 150 4.2.1 Experimental Groups 150 4.2.2 Virus Infection and Detection 151 vii 4.2.3 Gelatin Zymography 151 4.2.4 Western Blot 151 4.2.5 Histological Assessment 152 4.2.6 Immunohistochemistry 152 4.2.7 Quantitative Real Time Polymerase Chain Reaction (qPCR) 152 4.2.8 Data and Statistical Analyses 153 4.3 Results 153 4.3.1 Gelatinolytic Activation of M M P - 2 and M M P - 9 153 4.3.2 Protein Expression of M M P s 155 4.3.3 Morbidity and Histological Examination 158 4.3.4 hrimune Infiltration in K O and W T Mouse Hearts 160 4.3.5 Transcriptional Regulation o f Cytokines 164 4.4 Conclusions and Discussion 165 4.5 References 172 C H A P T E R V : N E U T R A L I Z I N G A N T I - 4 - 1 B B L T R E A T M E N T I M P R O V E S C A R D I A C F U N C T I O N I N V I R A L M Y O C A R D I T I S 177 5.1 Rationale 177 5.2 Materials and Methods 179 5.2.1 Virus Infection and Animal Treatment 179 5.2.2 Fluorescent Immunohistochemistry 179 5.2.3 Echocardiography 180 5.2.4 Histological Examination 181 5.2.5 R T - P C R 181 viii 5.2.6 Immunohistochemistjy 181 5.2.7 Data and Statistical Analyses 182 5.3 Results 184 5.3.1 Immunohistochemical Staining for 4-1BB and 4 - 1 B B L 184 5.3.2 Echocardiography 185 5.3.3 Histological Examination 189 5.3.4 Protease Expression 191 5.3.5 Immunohistochemical Staining for Immune Cells 192 5.4 Conclusions and Discussion 195 5.5 References 200 C H A P T E R V I : C O N C L U S I O N S A N D F U T U R E D I R E C T I O N S 203 A P P E N D I X 209 A . l List o f Publications, Abstracts, and Presentations 209 A . 2 Ethics Certificates 213 ix LIST OF TABLES Table 1.1: Causes of Myocarditis 4 Table 1.2: Picornaviridae Family of Viruses 7 Table 1.3: W H O A V o r l d Heart Federation Task Force for the Definition of Acute and Chronic Myocarditis Criteria 10 Table 1.4: Comparison of Results from T-cell Deficient Mice in V i r a l Myocarditis 28 Table 1.5: T N F Family Members: Co-Stimulatory Molecules Consisting o f Receptor/Ligand Pairs 29 Table 1.6: Matrix Metalloproteinase Family and Known Substrates 47 Table 1.7: Myocarditis Diagnosis and Treatment Strategies 61 Table 1.8: P C R Primer Sequences and Conditions for Detection of M M P s 92 LIST OF FIGURES Figure 1.1: Representative Images o f Coxsackievirus 8 Figure 1.2: Histological H & E Images of Human Heart Tissue 10 Figure 1.3: The Pathogenic Phases of Myocarditis and the Development of Cardiomyopathy 13 Figure 1.4: Early Vacuolar and Coagulative Myocyte Degeneration in Myocarditis 15 Figure 1.5: A Simplified Schematic Representation o f the Wound Healing Process 35 Figure 1.6: Affymetrix Oligonucleotide Microarray Analysis of Cathepsin Expression in CVB3-Infected Mouse Hearts 41 Figure 1.7: The Plasminogen System and Its Role in Remodeling 42 Figure 1.8: Archetypal Structures of M M P s 46 Figure 1.9: Outcomes of Myocarditis 54 Figure 1.10: Experimental Design 85 Figure 2.1: Affymetrix Microarray Analysis of M M P s 88 Figure 2.2: Plaque Assay Quantitation of Vi ra l Particles 97 Figure 2.3: Analysis of the Vi ra l Genome and Histological Assessment of the Myocardium by H & E and Picrosirius Red Staining 99 Figure 2.4: Transcriptional Analysis of M M P - 2 , M M P - 9 , and M M P - 1 2 100 Figure 2.5: Protein expression and localization of M M P s - 2 , -9, and 12 102 Figure 2.6: Immunofluorescent Co-Staining for CD45 and M M P s in Virus-Infected Hearts at 9 Days Post-Infection 104 Figure 2.7: Immunoblotting Analysis of M M P - 8 , M M P - 1 3 , and M T 1 - M M P 106 Figure 2.8: Zymography Analysis of M M P - 2 and M M P - 9 108 xi Figure 2.9: Immunoblotting Analysis of TIMPs in CVB3-Infected Hearts 109 Figure 3.1: Coxsackievirus Infection of HL-1 Cells 125 Figure 3.2: Expression of M M P - 1 2 and C V B 3 in Infected HL-1 Cells 127 Figure 3.3: Morbidity and Mortality Assessment o f CVB3-Infected M M P 1 2 K O and W T M i c e at 3 Days Post-Infection 129 Figure 3.4: Movat 's Staining of CVB3-Infected M M P 1 2 K O and W T Mice 132 Figure 3.5: Picrosirius Red Staining of CVB3-Infected M M P 1 2 K O and W T M i c e 134 Figure 3.6: Assessment of Virus Infection in M M P 1 2 K O and W T Mice 136 Figure 3.7: Apoptosis Analysis of CVB3-Infected M M P 1 2 K O and W T M i c e 138 Figure 4.1: Zymography Analysis of M M P - 2 and M M P - 9 Activities in CVB3-Infected M M P 9 K O and W T Mice 154 Figure 4.2: Translational Expression o f M M P s and T I M P s i n M M P 9 K O and W T Mouse Hearts 156 Figure 4.3: Morbidity Assessment of CVB3-Infected M M P 9 K O , M M P 8 K O , and Control M i c e at 9 Days Post-Infection 157 Figure 4.4: Analysis of Fibrosis in CVB3-Infected M M P 9 K O , M M P 8 K O , and Control M i c e at 9 Days Post-Infection 159 Figure 4.5: Immunohistochemistry Staining for CD45, Phagocytes, and T-cells in C V B 3 -Infected M M P 9 K O , M M P 8 K O , and Control Mice 163 Figure 4.6: Transcriptional Analysis of Cytokines in M M P 9 K O and W T M i c e 165 Figure 5.1: Co-Localization hnmunohistochemistry Staining for 4-1BB and 4 - 1 B B L in Sham and CVB3-Infected Mice 183 Figure 5.2: Echocardiography Analysis of Cardiac Function in Mice Following C V B 3 -X l l Infection 186 Figure 5.3: Echocardiography Analysis of Cardiac Function Following A n t i - 4 - l B B L Treatment in CVB3-Infected Mice 188 Figure 5.4: Histological Analysis of Morbidity and Fibrosis in CVB3-Infected M i c e Following A n t i - 4 - l B B L Treatment 190 Figure 5.5: P C R Analysis of M M P s - 2 , -9, and -12 in CVB3-Infected M i c e Following Ant i -4 - 1 B B L Treatment 192 Figure 5.6: Immunohistochemistry Analysis of CD45 and T-cells in CVB3-Infected M i c e Following A n t i - 4 - l B B L Treatment 194 Figure 6.1: Illustration of Hypothesized M M P Mechanisms During the Three Phases of Myocarditis 208 xiii LIST OF ABBREVIATIONS ACC American College of Cardiology ACE Angiotensin-converting enzyme ADAR Adenosine deaminase AHA American Heart Association APMA Aminophenylmercuric acetate cDNA Complementary deoxyribonucleic acid CHF Congestive heart failure CO Cardiac output CTL Cytotoxic lymphocytes CVB3 Coxsackievirus B3 DAB Diaminobenzidine DCM Dilated cardiomyopathy DMEM Dulbecco's modified Eagle medium ECM Extracellular matrix EF Ejection fraction EMB Endomyocardial biopsy ESETCID European Study on the Epidemiology and Treatment of Cardiac Inflammatory Disease FBS Fetal bovine serum GAPDH Glyceraldehyde-3-phosphate dehydrogenase HAART Highly Active Antiretroviral Therapy IFN Interferon xiv IgG Immunoglobulin type G IGTP IFN-Y inducible GTPase IL Interleukin IRF Interferon regulatory factor ISH In situ hybridization ivIG Intravenous immunoglobulin KO Knockout LPS Lipopolysaccharide LMCV Lymphocytic choriomeningitis virus LRP Low density lipoprotein receptor-related protein LV Left ventricular L VPW Left ventricular posterior wall M-CSF Macrophage colony stimulating factor MAP Mitogen-activated protein MHC Major histocompatibility complex MI Myocardial infarction MIP Macrophage inflammatory protein MMP Matrix metalloproteinase MOI Multiplicity of infection MV Mitral valve NIH National Institutes of Health NK cell Natural killer cell NO Nitric oxide X V NP-40 Nonidet P-40 NYHA New York Heart Association OAS Oligoadenylate synthetase PAI Plasminogen activator inhibitor PBS Phosphate buffered saline PFU Plaque forming units pi Post-infection PKR Protein kinase R PM Papillary muscle PMN Polymorphonuclear (cell) PREMIER Prevention of Myocardial Infarction early Remodeling PSLA Parasternal long axis RECK Reversion-inducing cysteine-rich protein with Kazal motifs rRNA Ribosomal ribonucleic acid ROS Reactive oxygen species RT-PCR Reverse transcriptase polymerase chain reaction SCID Severe combined immunodeficiency SD Standard deviation Serpin Serine protease inhibitor TACE TNF-OC converting enzyme TCR T-cell receptor TGF Transforming growth factor TIMP Tissue inhibitor of metalloproteinase TLR Toll-like receptor TNF (R) Tumour-necrosis factor (receptor) tPA Tissue-type plasminogen activator uPA(R) Urokinase-type plasminogen activator (recept VSV Vesicular stomatitis virus WHF World Heart Federation WHO World Health Organization WT Wildtype A C K N O W L E D G E M E N T S These past 6 years have been life-altering and exceptional not only because of all the knowledge and experience I have gained as a researcher but I am also enriched by all the people I have met and interacted with at the i C A P T U R E Centre. A l l the members, past and present, o f this centre are amazing scientists and friends; their diligence, intelligence, passion for science, and ardent support of others are attributes that I hope to foster in myself. I would like to begin by thanking my mentor Dr. Bruce McManus for giving me the opportunity to work under his supervision. His passion for science, respect for others, and conscientiousness to research, the community, and human health truly make him a great role model. I w i l l always remember his teachings and I hope his mentorship does not end with my dissertation, for I still have a lot to learn from him. I also enjoyed mentorship from very special individuals in our laboratory. I would like to thank Dr. Honglin Luo for her support through all o f these years, her kindness and friendship truly helped me through the many obstacles I encountered. I sincerely admire her integrity, meticulousness, logical experimentation, and her focus. Another colleague who has been instrumental in my development as a scientist is Dr. Bobby Yanagawa. He is a great friend and mentor to me and I would like to thank him for all o f his help, which he continues to give even after he has left the laboratory. I truly admire his passion for knowledge and his time management skills. I would also like to thank all o f the exceptional members at the i C A P T U R E Centre, in particular Dr. Jingchun Zhang, M s . Elizabeth Walker, M s . Zongshu Luo, Dr. Hon Sing Leong, Dr. Maziar Rahmani, Dr. Hubert Walinski , and Dr. J i Yuan for their guidance and assistance, both technically and mentally. I really appreciate the assistance from the animal care technicians at Jack Be l l Centre and G E M facility, for all xviii of my projects would not have been possible without their aid. I am also very grateful to my supervisory committee members, Drs. Keith Walley, Edwin Moore, Ed Pryzdial, and Christopher Overall, for their enlightening discussions and valuable advice on my research. Dr. David Walker has also been a great friend and teacher; beginning from the Direct Studies course I took with him in my first year, he continues to nurture and advise me in my studies. In addition, my projects are only possible due to collaboration with other scientific groups; thus I would like to extend my appreciation to Drs. Overall and Robert Senior for providing the M M P - 8 and M M P - 9 knockout mice, respectively, and Dr. Tracy Deisher, formerly at Amgen Corporation. She has shown me that a female scientist can balance work and family life wel l , and that practice does make perfect. Further, I would like to express my deepest gratitude to the scientific funding agencies which sponsored my research. In particular, the Canadian Institutes o f Health Research (CIHR) have provided the majority of operating funds and conferred a doctoral fellowship to me. Last but not least I would like to thank the people who have supported me for all or the greater part of my life. I really appreciate the love and generosity that my parents, Y u n Fong and Siu K i n g Cheung, and my siblings, Hilton and Teresa Cheung, have shown me and the sacrifices they made to support me. I am also grateful for wonderful friends, especially Christine Chung, Lana Chow, and Dr. Hisashi Suzuki, who have grown with me and helped me through difficult times. The James Hogg i C A P T U R E Centre is truly an exceptional institute: the multicultural atmosphere mirrors the interdisciplinary research interests where the science is o f the highest grade due to the liberality and multilayered collaborations between the investigators. xix C O - A U T H O R S H I P S T A T E M E N T I performed the majority of the experiments but they were made possible because of many collaborative efforts with various researchers. M s . Zongshu Luo, Dr. Jichun Zhang, and M s . Elizabeth Walker aided me in many molecular techniques, such as R N A extraction, R T - P C R , plaque assay, cell culture, and immunohistochemistry. M r . John La i and M r . Hon Sing Leong instructed and guided me in immunohistochemistry and confocal microscopy, respectively. I also received aid in histological staining from various investigators: M s . Agripina Suarez helped me perform in situ hybridization, M s . Amri t Samra aided in histological sectioning and staining, and M s . Hongyan Zhao performed T U N E L staining for apoptosis. I was trained to use echocardiography in mice by M s . Stefanie Bonigut and Dr. Theresa Deisher, who also provided direction and consultation in experimental design and execution. Dinesh Samarasekera was a summer student who, under my instruction, investigated the role of M M P - 1 2 in HL -1 cells. Dr. Honglin Luo was instrumental in instruction and implementation of the zymography technique. Both Dr. Luo and Dr. Bobby Yanagawa were major collaborators and guided me in data acquisition, critical analyses of my results, and manuscript preparation. Lastly, Dr. Bruce McManus provided much guidance, support, and input on data analyses, manuscript preparation, as well as overall project planning and implementation. XX CHAPTER I: INTRODUCTION TO VIRAL MYOCARDITIS The overall aim of this dissertation is to better understand the processes instigated in the host to combat viral infection and to repair the myocardium after injury. A well -established murine myocarditis model, caused by coxsackievirus B3 ( C V B 3 ) infection, is used because the pathogenesis and progression of disease in this animal model are very similar to that observed in humans. Myocarditis has important clinical relevance in the human population and despite extensive research in this area; the pathogenesis of this disease has still not been fully elucidated. 1.1 Overview of Myocarditis In the early 19 century, myocarditis was initially recognized by Sobernheim [1] and then in 1899, Kar l Fiedler [2] first used light microscopy to describe a myocardial disorder that was characterized by immune infiltration in the interstitial tissue, swelling o f the myocytes, decay of crossbands, and areas of necrotic muscle fibers. Since then, this description has withstood the test o f time and is still used today, albeit with some modifications. Myocarditis generally causes a decrease in left ventricular ( L V ) myocardial function due to loss of viable myocardium and can occur in all age groups, albeit fatality in infants is much higher than adults. Myocarditis is the major cause of sudden unexpected death in patients <40 years o f age who were otherwise healthy and examination of a large, random series of autopsy myocardial samples indicated that 1-5% of the general population and 5-12% of sudden death in young adults suffer from undiagnosed myocarditis [3]. The statistical information for frequency of myocarditis in the human population varies amongst published data and is difficult to obtain 1 because sub-clinical symptoms, varying diagnosis, and unpredictability of the prognosis result in underestimation of diagnosed cases and delayed treatment [4]. Congestive heart failure (CHF) and dilated cardiomyopathy ( D C M ) are believed to result from myocarditis [5]. A s yet, no definitive therapy has been shown to be effective in treating myocarditis and often the end result is heart transplantation, where recurrence of myocarditis and transplant rejection can occur. 1.2 Definition of Myocarditis Myocarditis is a non-ischemic inflammatory disease of the myocardium, of which there are a few common types: lymphocytic, eosinophilic, neutrophilic, granulomatous, and giant cell myocarditis [6,7]. In the first case, the immune infiltrate consists predominantly o f mononuclear cells whereas in giant cell myocarditis, diffuse, non-granulomatous inflammation consisting of abundant multinucleated giant cells is observed. The established histological diagnosis of myocarditis is through classification of right ventricular endomyocardial biopsy ( E M B ) specimens into three types as defined by the Dallas Criteria (1986): active, borderline, and nonmyocarditis [8]. Active myocarditis is characterized by presence of immune cells and myocytolysis within the myocardium that is not associated with ischemic events. Borderline disease is defined as less intense immune infiltration and degeneration of myocytes not seen with standard light microscopy. Nonmyocarditis cases have none or rare immune cells in the myocardium. Lieberman and colleagues [9] proposed a broader clinicopathological classification of myocarditis which combines clinical features of the disease to the histological presentations to extend the definition of myocarditis beyond the narrow confines of the Dallas criteria. This classification divides myocarditis into 2 fulminant, acute, chronic active, and chronic persistent subtypes. Patients with fulminant myocarditis are presented with severe illness, including significant hemodynamic deterioration and myocardial dysfunction, which may be fatal or may spontaneously resolve, and the diagnosis can be confirmed with histological evidence of immune infiltration in the E M B samples. Acute myocarditis has less distinct clinical presentations compared to the fulminant type and is defined as modest cardiac dysfunction with gradual deterioration over time. Histological examination usually shows immune infiltration at time o f presentation, which gradually resolves but the patient may develop D C M over time. Chronic active myocarditis is similar to acute myocarditis at first presentation, when the patient may experience modest cardiac dysfunction and histological examination confirms active myocarditis. However, this type is also characterized by ongoing inflammation, interstitial fibrosis, and development of cardiomyopathy. Lastly, chronic persistent myocarditis is characterized by atypical chest pains or palpitations, but with no obvious signs or symptoms of myocardial dysfunction, and ongoing inflammation in the myocardium. Although the categorization by Lieberman's group greatly enhances the Dallas criteria, this classification is seldom used. Currently, the term "inflammatory cardiomyopathy", which was first introduced in 1995 by the World Health Organization/World Heart Federation ( W H O / W H F ) Task Force on Definition and Classification of Cardiomyopathies to define "myocarditis associated with cardiac dysfunction", is used most frequently [10]. 3 1.3 Etiology of Myocarditis Myocarditis can develop following many different cardiac injuries stemming from viral, bacterial, fungal, and parasitic infections as well as hypersensitivity, drug-induction, and autoimmunity, some of which are shown in Table 1.1. Table 1.1: Causes of myocarditis (Adapted from Feldman and McNamara [11]). Etiological agents of myocarditis are demarcated into three main types based on their biochemical nature: infectious, immune-mediated, and toxin-related. The most common cause of infectious myocarditis is coxsackievirus (bold-faced in Table). INFECTIOUS IMMUNE-MEDIATED TOXIN-RELATED Bacterial Brucella, Corynebacterium diphtheriae, Gonococcus, Haemophilus influenzae, Meningococcus, Myocobacterium, Mycoplasma pneumoniae, Pneumococcus, Salmonella, Serratia marcescens, Staphylococcus, Streptococcus pneumoniae, Strep, pyogenes, Treponema pallidum, Tropheryma whippelii, Vibrio cholerae Allergens Acetazolamide, Amitriptyline, Cefaclor, Colchicine, Furosemide, Isoniazid, Lidocaine, Methyldopa, Penicillin, Phenylbutazone, Phenytoin, Reserpine, Streptomycin, Tetanus toxoid, Tetracycline, Thiazide Drugs Amphetamines, Anthracyclines, Catecholamines, Cocaine, Cyclophosphamide, Ethanol, Fluorouracil, Hemetine, Interleukin-2, Lithium, Trastuzumab Spirochetal Borrelia, Leptospira Alloantigens Heart-transplant rejection Heavy Metals Copper, Iron, Lead Fungal Actinomyces, Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Histoplasma, Mucormycoses, Nocardia, Sporothrix Autoantigens Chagas' disease, Chlamydia pneumoniae, Churg-Strauss syndrome, Inflammatory bowel disease, Giant-cell myocarditis, Insulin dependent diabetes mellitus, Kawasaki's disease, Myasthenia gravis, Polymyositis, Sarcoidosis, Scleroderma, Systemic lupus erythematosus, Thyrotoxicosis, Wegener's granulomatosis Physical Agents Electric shock, Hyperpyrexia, Radiation Protozoal Toxoplasma gondii, Trypanosoma cruzi Miscellaneous Arsenic, Azides, Bee/wasp stings, Carbon monoxide, Inhalants, Phosphorus, Scorpion bites, Snake bites, Spider bites Parasitic Ascaris, Echinococcus granulosus, Paragonimus westermani, Schistosoma, Taenia solium, Trichinella spiralis, Visceral larva migrans, Wuchereria bancrofti Rickettsial Coxiella burnetii, Rickettsia rickettsii, Rick, tsutsugamushi Viral Coxsackievirus, Cytomegalovirus, Dengue virus, Echovirus, Encephalomyocarditis, Epstein-Barr virus, Hepatitis A virus, Hepatitis C virus, Herpes simplex virus, Herpes zoster, Human immunodeficiency virus, Influenza A virus, Influenza B virus, Parvovirus, Poliovirus, Rabies virus, Respiratory syncytial virus, Rubella virus, Rubeola, Vaccinia virus, Varicella-zoster virus, Variola virus, Yellow fever virus 4 Depending on the causative agent, myocarditis has different manifestations upon examination o f hemodynamic parameters and the amount, distribution, and subtypes o f immune cells. For example, a rare and frequently fatal type of myocarditis that predominantly strikes young, healthy adults is giant cell myocarditis. This disorder is suggested to have non-infectious origins, since detection o f infectious particles from diagnosed sample cases have been unsuccessful, and it is often associated with autoimmunity, such as chronic hepatitis, systemic lupus erythematosus, inflammatory bowel disease, Takayasu's arteritis, rheumatoid arthritis, optic neuritis, and type I diabetes [6]. The clinical presentation of patients suffering from this form of myocarditis usually include exertional dyspnea, decreased exercise tolerance, peripheral edema, and rapid onset o f cardiovascular dysfunction, such as arrhythmia and tachycardia, which may lead to C H F and sudden death. The histological characteristics usually consist of varying degrees of fibrosis and non-granulomatous immune infiltration, where lymphocytes, granulocytes, histiocytes, and profuse multinucleated giant cells are frequently observed. The prognosis is usually grim for giant cell myocarditis where rate o f death or heart transplantation is approximately 70% at 1 year following diagnosis [12]. Hypersensitivity to various drugs, including antidepressants, antibiotics, and antipsychotics may result in eosinophilic myocarditis. Symptoms include fever, peripheral eosinophilia, sinus tachycardia, drug rash, and immune infiltration into the myocardium, consisting predominately of eosinophils with some lymphocytes. This form of myocarditis usually is not fulminant, is generally reversible, and subsides with cessation o f drugs or administration of corticosteroids [4,13]. 5 A major emerging form of myocarditis is seen in HIV-infected patients. This type of myocarditis is increasing alarmingly rapidly due to greater number o f cases of H I V infection; in 2003 alone, approximately 40 mil l ion people were infected and 3 mil l ion deaths were recorded [14]. Autopsy documentation has revealed a 40-50% frequency o f myocarditis in HIV-infected patients [15,16]. Upon examination of histological samples from HIV-infected patients, diffuse immune infiltration was detected in the myocardium, consisting mainly of lymphocytic infiltrates but the degree of inflammation does not always correlate with severity of cardiac symptoms [16,17]. The cardiovascular symptoms are usually overshadowed by the underlying condition of the H I V patient, and histological confirmation of myocarditis is usually conducted after spontaneous L V dysfunction has occurred [16,18]. Using in situ hybridization, the H I V genome has been detected within cardiomyocytes [17,19]. However, the mechanism of myocarditis induction is still unclear but it has been postulated that hypersensitivity to anti-HIV medications, H I V infection itself, and/or opportunistic myocardial co-infection associated with toxoplasmosis, tuberculosis, cryptococcosis, histoplasmosis, aspergillosis, cytomegalovirus infection, herpes simplex virus infection, or coxsackievirus B3 infection may be responsible for induction o f myocarditis [14,20-23]. In industrialized countries, the administration of highly active antiretroviral therapy ( H A A R T ) to HIV-infected patients has greatly decreased the incidence o f myocarditis and other cardiomyopathies associated with A I D S but in developing countries, lack of proper medication and medical help has decreased the availability of this treatment to H I V patients. Table 1.2: Picornaviridae family of viruses (Adapted from Stanway et al. [24]). The current classification of Picornaviruses are listed below but due to more detailed investigations of their genomes, it has been proposed that the rhinoviruses be combined with the enterovirus group and a novel genera, the sapelovirus group, be introduced. Currently, the enterovirus group includes 20 C V A and six C V B subtypes. CURRENT GENERA PROPOSED GENERA Enterovirus "|* Enterovirus Rhinovirus Cardiovirus Cardiovirus Aphthovirus Aphthovirus Heptovirus Heptovirus Parechovirus Parechovirus Erbovirus Erbovirus Kobuvirus Kobuvirus Teschovirus Teschovirus Sapelovirus CVA CVB C V A 1 C V B 1 C V A 2 C V B 2 C V A 3 C V B 3 C V A 4 C V B 4 C V A 5 C V B 5 C V A 6 C V B 6 C V A 7 C V A 8 C V A 9 C V A 1 0 CVA11 /15 C V A 12 C V A 1 3 / 1 9 C V A 1 4 C V A 16 C V A 1 7 C V A 1 8 C V A 2 0 C V A 2 1 C V A 2 2 7 Figure 1.1: Representative images of coxsackievirus. Transmission electron micrograph (left) [Adapted from Dr. Stewart M c N u l y et al., Veterinary Sciences, The Queen's University of Belfast] and in sz/zco-generated representation (right) [Adapted from Dr. Thomas Ferrin et al., Resource for Biocomputing, Visualization, and Informatics; University of California, San Francisco] o f coxsackievirus are shown. Ultrastructural imaging and biochemical analyses, using techniques such as X-ray crystallography, of coxsackievirus indicate that these particles are 28-30nm in diameter and icosahedral in shape. The W H O reports that the incidence of cardiovascular disorder after enteroviral infection is 1-4%, depending on the causative organism. The Picornoviridae family and Enterovirus genus include echovirus, polioviruses, and coxsackieviruses (CV) , as shown in Table 1.2 [24]. Coxsackievirus is grouped into two types: C V A , which consists of 20 serotypes, and C V B , which includes 6 serotypes. C V are non-enveloped viruses with a hexagonal external structure, approximately 30nm in diameter, and a single positive strand ribonucleic acid ( R N A ) genome, approximately 7.4 kilobases in size (Figure 1.1). C V group A viruses belong to a large family ( A l to A24) but previous classifications have changed slightly to reflect new evidence that showed that some of them are indeed the same strain ( A l l is identical to A15 and A13 is identical to A19) while two members, A23 and A24 , have recently been revealed to be echoviruses [25]. C V A infections are generally associated with hand, foot, and mouth disease, characterized by vesicular lesions in these areas, particularly 8 in young children and infants [26]. Other clinical presentations include herpangina, asceptic meningitis, petechial or vesicular rashes, colds, and acute hemorrhagic conjunctivitis. C V A usually does not cause myocarditis except C V A 16 has been detected in patients presenting with myocarditis [27]. The major clinical manifestations o f C V B infections consist o f myocarditis, pericarditis, neonatal systemic disorders, aseptic meningitis, meningoencephalitis, acute flaccid paralysis, herpangina, and potentially type 1 diabetes [28,29]. Recent reports by the National Enterovirus Surveillance System have shown that each year, 10-15 mil l ion symptomatic enteroviral infections are documented worldwide and of the cases with known serotypes, approximately 25% are caused by the C V B viruses [25]. Epidemiological records show that C V B 3 and C V B 4 are the predominate causes of myocarditis and these viral genomes were consistently detected in autopsy and biopsy samples as well as explanted hearts [30-32]. The most prevalent characteristics of coxsackievirus-induced myocarditis in humans are lymphocytic infiltration with necrotizing myocytes, showing signs of irregular contraction banding patterns, vacuolization, cytoplasmic swelling, and fragmentation of nuclei (Figure 1.2). However, as described above, the clinical presentations are often diverse and infected individuals frequently experience rather mild symptoms, such as fever, fatigue, myalgia, diarrhea, dyspnea on exertion, palpitations, and malaise [3]. Chest discomfort, including pleuritic, sharp, stabbing pain or substernal and squeezing pain, is often reported in patients [33]. These subclinical cases may also involve transient E C G abnormalities. Pediatric patients, particularly infants, also exhibit nonspecific symptoms: fever, respiratory distress, poor feeding, and cyanosis in severe cases [34]. 9 Figure 1.2: Histological H&E images of human heart tissue. Normal human heart, shown on the right, is devoid of infiltrating immune cells whereas extensive inflammation is detected in CVB3-infected human heart with active myocarditis (right). Table 1.3: WHOAVorld Heart Federation Task Force for the definition of acute and chronic myocarditis criteria [36]. In 1995, the W H O / W H F on the Definition and Classification of Cardiomyopathies introduced several changes in the definition and classification of myocarditis, thus refining the Dallas classification. FIRST BIOPSY Acute (active) myocarditis: A clear-cut infiltrate (diffuse, focal or confluent) o f >14 leukocytes/mm 2 (preferably activated T-cells). The amount of the infiltrate should be quantitated by immunohistochemistry. Necrosis or degeneration are compulsory, fibrosis may be absent or present and should be graded. Chronic myocarditis: A n infiltrate o f >14 leukocytes/mm 2 (diffuse, focal or confluent, preferably activated T-cells). Quantification should be made by immunohistochemistry. Necrosis or degeneration are usually not evident, fibrosis may be absent or present and should be graded. No myocarditis: No infiltrating cells or <14 leukocytes/mm 2. SUBSEQUENT BIOPSIES Ongoing (persistent) myocarditis: Criteria as in 1 or 2 (features of an acute or chronic myocarditis). Resolving (healing) myocarditis: Criteria as in 1 or 2 but the immunological process is sparser than in the first biopsy. Resolved (healed) myocarditis: Corresponds to the Dallas classification. 10 1.4 Diagnosis The standard diagnostic tool for verification of suspected myocarditis is examination of right ventricular E M B samples and classification of disease as implemented by the W H O / W o r l d Heart Federation Task Force for the Definition o f Acute and Chronic Myocarditis in 1996 [35], as outlined in Table 1.3. This is a modification o f the Dallas criteria and includes more specific details of the amount of infiltrates: more than 14 leukocytes/mm 2 o f cardiac tissue consisting predominately of activated T-cells, as assessed by immunohistochemistry, define active or chronic myocarditis [35,36]. However, there are many issues with the diagnostic procedure, sampling, and interpretation. The current American College of Cardiology/American Heart Association ( A C C / A H A ) guidelines indicate that endomyocardial biopsies are not included in the routine evaluation of cardiomyopathy except in cases where the suspected disorder is supported by additional data and only when there is "a strong reason to believe that the results w i l l have a meaningful effect on subsequent therapeutic decisions or prognosis" [37]. Due to the varying nature o f myocarditis, patients are often ineligible for the biopsy procedure because they are usually asymptomatic or the disease may be masked by or masquerades as other disorders, such as A I D S or myocardial infarction (MI), respectively [17,38]. El igibi l i ty for E M B does not guarantee accurate diagnosis. Sampling o f the affected areas is necessary in order to obtain cardiac tissue that is representative of the disease. However, biopsies are usually taken in the right ventricular wall o f the intraventricular septum and myocarditis is characterized by diffuse, focal inflammation rather than large, regional injuries so sampling error results in false negatives when obtaining biopsy samples [39]. For example, in one study performed in Finland, of 142 autopsy specimens with 11 suspected myocarditis, only 32% had histological confirmation using the Dallas criteria [40]. In our own laboratory, previous findings show that less than 50% of the first biopsies were diagnosed with myocarditis [41]. This suggests that multiple biopsies should be performed to not only chart the disease progression but also to ensure appropriate sampling and proper diagnosis. Further, interobserver variability in interpretation of the biopsy samples is very great, despite high levels o f expertise of the pathologists. For example, in the Myocarditis Treatment Trial , only 64% of the 111 patients, who were originally diagnosed with myocarditis by endomyocardial biopsy, were confirmed by the expert pathology panel who further reviewed the histological specimens [8,42]. In a separate study, assessment of endomyocardial biopsies of 16 patients with D C M by seven expert pathologists varied widely in the diagnosis o f myocarditis as well as other histological features [43]. Definite or probable myocarditis was diagnosed in 11 of 16 patients by at least one pathologist, however, less than 50% of the pathologists agreed upon any one of the diagnosed cases. Recent progress in other diagnostic tools, utilizing molecular biological techniques (reverse-transcriptase P C R [RT-PCR] and in situ hybridization [ISH]), biomarker assessment (immunohistochemical detection of inflammatory markers [CD3, CD45, M H C ] and serum detection o f cardiac markers [troponin I and T]), and non-invasive imaging techniques have emerged to improve the diagnosis of myocarditis [4,44]. The use of R T - P C R and ISH for detection o f microbial genomes can aid in the diagnosis of infectious myocarditis i f the detected agent is known to cause myocarditis, such as C V B [44,45]. The presence o f infectious particles in the patients' samples can serve as supportive data for eligibility for the E M B procedure. Also , to decrease interobserver variability on the interpretation of amount 12 of immune infiltration, necrosis, and fibrosis, quantitation of immunostaining for inflammatory markers, such as CD3 for T-cells and CD45 for pan-leukocytes, and the complement protein, C5b-9 as well as picrosirius red staining for collagen would produce more objective results [4,44]. However, to eliminate the use of endomyocardial biopsies, the use of non-invasive imaging may be effective. Echocardiography, tissue-doppler, gallium-67 imaging, i n d i u m - I l l anti-myosin scintigraphy, and magnetic resonance imaging have all been used successfully in some cases for diagnosis of myocarditis [4,39,46]. Figure 1.3: The pathogenic phases of myocarditis and the development of cardiomyopathy. V i ra l entry and viremia occurs until 3 days pi , followed by the inflammatory phase characterized by massive immune infiltration. Reclamation and remodeling of the damaged myocardium is usually regarded as the chronic phase of the disease since these events may continue for months and years following the initial infection, possibly leading to D C M and heart failure through maladaptive remodeling. Days Post- Infection 0-3 4-14 >15 Months-Years V ira l Immune Infiltration Reclamation of the Heart Dilation of Heart 13 1.5 Pathogenesis The most common and well-established model of myocarditis is coxsackievirus B3 ( C V B 3 ) infection of adolescent mice, which induces a disorder that is histopathologically similar to the human disease. This model produces a disease involving three distinct phases: viremic (0-3 days), inflammatory (4-14 days), and resolution/remodeling (>15 days), as pictorially depicted in Figure 1.3. The evolution of the disease results in D C M within months to a year following the initial virus infection [47]. During the first phase of the disease, viral genome and antigen can be detected within the circulation and myocardium, leading to direct virus-mediated cardiac injury and myocytolysis [48]. The cardiomyocytes exhibit distinct contraction band patterns, swelling of the cytoplasm, vacuolization, and fragmentation of nuclei, as shown in Figure 1.4. A t this timepoint, no or minimal inflammatory cells are detected within the myocardium. Extensive immune infiltration enters the myocardium beginning from 5 days post-infection (pi). The innate immune system is initially activated following infection and the first wave of immune cells consists of mostly natural killer ( N K ) cells and macrophages followed by the lymphocytes of the acquired immune system [49]. The innate immune cells first aid in clearance of the virus in infected cells and they also mediate cytolysis o f infected cells. The T-lymphocytes are activated to produce CD8+ or cytotoxic lymphocytes ( C T L ) and CD4+ helper cells in order to further eliminate infected cells and to produce anti-virus antibodies, respectively. During this phase of the disease, immune-mediated injuries to the myocardium can be observed and chronic damage to the heart may occur through inappropriate lymphocytic functions, such as cytolysis o f uninfected cardiomyocytes by C T L activities and production of heart-specific auto-antibodies mediated by the CD4+ T-cells. B y 14 days pi , inflammation within the heart has subsided and reparation 14 and resolution o f the myocardium is proceeding. Following extensive myocytolysis from the initial injuries, replacement fibrosis occurs, which is characterized by abundant collagen accumulation within the myocyte dropout regions, as well as interstitial or reactive fibrosis, which is independent of myocyte loss and in areas of viable tissue. The architecture and organization of the collagens within these areas are not properly formed and remodeling of these scars is necessary in order for proper alignment of cardiomyocytes and vasculature so that cardiac structure and function is maintained. Several factors influence this remodeling and reparation process: the degree of injury or amount of viable tissue remaining, persistence o f virus and inflammation, and the balance of matrix regulators, such as myofibroblasts and extracellular matrix ( E C M ) proteases (matrix metalloproteinases or M M P ) that degrade matrix components [47,50]. Figure 1.4: Early vacuolar and coagulative myocyte degeneration in viral myocarditis. This image shows a section of an infected murine myocardium and the arrow indicates a potentially infected myocyte undergoing necrosis, characterized by vacuolization. 15 1.5.1 Direct Virus-Mediated Injury During the initial phase o f myocarditis, the virus enters the circulation and infects ventricular cardiomyocytes. The time course of infection and mechanism o f cytolysis has been studied in vitro in human and mouse cell lines [51-53]. During the replicative and virus dissemination process, cytopathic effects, which include nuclear shrinkage, condensation o f chromatin, cell rounding, acidophilic cytoplasm, nuclear pyknosis, and fragmentation of D N A , occurs and the host cell is injured, eventually leading to cell death [53]. C V B 3 viral proteases have been shown to cleave the host's structural and transcriptional proteins to modify endoplasmic reticulum and cell membrane permeability, causing an increase i n the cytosolic free calcium concentration and membrane ruptures which leads to the collapse of ionic gradients and ultimately host cell death [54,55]. There are two types of cell death, necrosis and apoptosis, and the relative contributions o f these two processes to cardiomyocyte cytolysis during myocarditis are still a matter of debate. The most prevalent method of myocytolysis is necrosis, leading to release of cytoplasmic constituents capable of triggering the early acting complement components. The complement system is activated v ia non-specific recognition o f pathogens and formation of proinflammatory components amplifies both the innate and adaptive immune responses [56]. Necrosis also generates reactive oxygen species (ROS), which have the potential to directly injure cardiac cells and may upregulate cytokines and chemokines to stimulate the immune response [57,58]. Despite low levels of apoptosis in myocarditis, this form of cell death may still play an important role. D C M patients exhibit signs o f apoptosis within the myocardium and in C V B 3 -infected mice suffering from myocarditis, apoptotic cells are also detectable in inflammatory lesions as well as in adjacent myocardial tissue [59-62]. During the early phase o f infection, it 16 would be advantageous for the virus to inhibit host cell death to maximize viral progeny production but at late stages of the viral life cycle, induction of apoptosis rather than necrosis for progeny release aids in evasion of the host immune surveillance system, thus increasing secondary infection of neighbouring cells. In apoptosis, infected cells are rapidly phagocytosed without the release of proinflammatory cytokines and intracellular components, which can cause recruitment and activation of immune cells [63]. Viruses can regulate the apoptotic pathway via several mechanisms: 1) interaction with pro-apoptotic proteins such as Siva, which is involved in CD27/CD70-transduced apoptosis; 2) activation of caspases leading to cleavage o f host substrates and mitochondrial dysfunction; 3) direct cleavage of I K B O I leading to inhibition o f N F - K B transactivation and downregulation o f survival genes; and/or 4) increase expression of the E C M protein Cyr61, leading to JNK-media ted activation o f apoptosis [64-67]. The outcome of uncontrollable viral replication and dissemination is extensive myocytolysis, degeneration of myofibers leading to alteration in electrophysiological processes, hypertrophy of the viable myocardial tissue, L V dysfunction, and persistence o f the virus which may lead to longterm damage to the heart. Therefore, a balanced and properly regulated host immune system is crucial in clearance of virus and infected cardiomyocytes as well as regulation of reparation and remodeling of the injured myocardium. While there is general agreement that direct virus-mediated cytolysis mediates progression o f myocarditis, the role o f the immune system is still under debate. 17 1.5.2 Immune Response 1.5.2.1 Innate Immunity The host immune response to virus infection consists of the innate immunity and the acquired/adaptive immune system. The first immune cells to be activated following a viral attack are the N K cells and phagocytes, which play very important roles as the first line of defense against virus infection and clearance. Previous studies showed that virus-infected mice treated with N K cell-blocking antibodies had increased severity of myocarditis as a result of increased viral replication and release [68,69]. Following CVB3-infection in N K cell-depleted mice, there was no significant difference in the number of inflammatory and scar lesions but these lesions exhibited increased dystrophic calcification and deterioration [69]. In human myocarditis tissue samples, the infiltration of N K cells into the myocardium is variable, depending on the form and stage o f myocarditis [70-72]. This immune subtype has cytotoxic capabilities similar to C T L and can eliminate infected cells, however, the anti-viral mechanism of N K cells is still not clear but may involve perforin activity but not the interferon (IFN) system [68,73,74]. Recent investigations have shown the importance o f toll-like receptors (TLR) , which also belong to the innate immune system and recognize non-specific foreign antigens, in viral clearance by recognition o f viral antigens and genome within the infected cell , leading to upregulation of cytokines to recruit and activate immune cells [75,76]. T L R 4 have been detected on mast cells and macrophages in viral myocarditis and this suggests that activation o f these cell-surface molecules may additionally lead to recruitment and activation of N K cells [77]. 18 Mast cells have also been detected during the early stages of myocarditis in mice as well as in active myocarditis and D C M patients [78,79]. However, the role of mast cells in myocarditis has not been extensively studied. One study showed that degranulation of activated mast cells secreted cytokines such as T N F - a , IL-1 (5, and IL-4 within hours of C V B 3 infection of mice [80]. Additionally, mast cells immediately upregulate expression o f T L R 4 following C V B 3 infection, a process that may be modulated by IFN-y and IL-10 [81,82]. Macrophages constitute one of the largest immune cell populations in myocarditis and they are observed very early in the infection and often still detected during the chronic stage of the disease [83]. During the early phase of the infection macrophages have phagocytic functions and may play a defensive role as a nonspecific immune defense mechanism against viruses. Administration o f macrophage colony stimulating factor (M-CSF) , which increased monocyte and macrophage counts in the circulation and heart, to CVB3-infected mice decreased morbidity and mortality by limiting myocardial virus titers through upregulation of I F N - a in acute C V B 3 myocarditis [84]. Further, investigation o f nitric oxide (NO) in C V B 3 -infected mice showed that N O is produced by macrophages and is a cytotoxic mediator o f this cell type. Depletion o f N O by treating mice with N O synthase inhibitors increased morbidity, mortality, and titers o f virus in treated mice [85,86]. However, the role of macrophages in myocarditis is debatable since several other studies show contrasting results. Some evidence suggests that macrophages are specifically involved in CVB3-induced myocarditis by maintaining a chronic inflammatory response. Studies that deplete macrophage-recruiting chemokines, such as macrophage inflammatory protein-la ( M l P - l a ) and MIP-2, result in a reduction in cardiac injury and inflammation as well as an increase in the survival rate but with no significant difference in viral titers [87,88]. Additionally, C V B 3 infection of freshly 19 isolated human monocytes induced activation of monocytes as evidenced by enhanced adherence, release of cytokines ( T N F - a , I L - l , and IL-6), and secretion of prostaglandin E2 [89,90], all o f which prolong the inflammatory response by further recruitment and activation of immune cells. The difference in results from these studies may be explained by the stage o f the disease, where early macrophage activity may be beneficial to the infection by phagocytosis o f infected cells and clearance of virus thus circumventing further infection. However, chronic infiltration and activation of macrophages may prolong the inflammatory response, leading to chronic myocarditis and deterioration of the heart. Other immune subtypes of the innate system, such as the granulocytes (neutrophils, eosinophils, and basophils), have not been studied extensively in the setting o f viral myocarditis because the frequencies of these immune subtypes in viral myocarditis is usually quite low. In some cases, such as hypersensitivity to certain medications resulting in allergy-related myocarditis, abundant eosinophil infiltration is observed, as described above. Although neutrophils or P M N s may constitute only a small portion of immune cells in viral myocarditis, their role against viruses may still be important. A recent study suggests that P M N s in C V B 3 -infected mice may act to decrease viral replication, thereby decreasing morbidity and development o f chronic myocarditis [91]. P M N s synthesize many modulating factors, such as cytokines, growth factors, and proteases which are stored within granules and are secreted upon immune activation to recruit, activate, and induce proliferation of immune cells [92,93]. The innate immune cells generally decrease virus replication through their cytotoxic or phagocytic capabilities but they usually work in conjunction with and are regulated by interferons. IFNs are a family of anti-viral molecules and they are classified into two types: types I ( IFN-a , IFN-p\ IFN-e, I F N - K , IFN-5, I F N - T , and IFN-co) which bind to IFN-oc receptors 20 and type II (IFN-y) binds to IFN-y receptors [94,95]. There is a single copy o f each I F N gene, except IFN-a , which includes 13 separate genes. During a viral infection, the predominant IFNs expressed are I F N - a , IFN-p, and IFN-y, and the ratio o f IFNs produced by cells varies with the tissue of origin, the virus strain, and the nature of viral challenge [96]. In general, type I IFNs are produced by virus-infected cells as part of the initial anti-viral response whereas IFN-y is produced upon mitogenic or antigenic stimuli during the delayed response [97,98]. IFNa/p are usually associated with stimulation of an anti-viral state within the infected cell in an autocrine and paracrine manner as well as stimulation of the innate immunity. IFN-y modulates both the innate and the adaptive responses by stimulation o f innate cells, such as N K cells and macrophages, as well as stimulation o f lymphocytes based on the recognition of cell surface-bound viral antigens expressed in association with major histocompatibility complex ( M H C ) proteins. Upon infection by virus, IFNs activate various signalling pathways, including the mitogen-activated protein ( M A P ) kinase complex, J N K , Jak-STAT, and p38, leading to activation of multiple transcription factor families, such as N F - K B , ATF-2/c-Jun, and interferon regulatory factors (IRFs) [99-102]. A s viral transcription and replication progress in the infected cell, viral products, including the viral capsid proteins and the formation of double-stranded R N A (which is the hallmark of a viral infection), become cofactors for activation of the intracellular anti-viral signaling complexes and the antigenic triggers for the innate immune response. IFNs induce intracellular effector proteins such as the R N A -activated protein kinase P K R , protein GTPase M x , 2'-5-oligoadenylate synthetase (OAS) , and adenosine deaminase ( A D A R 1 ) to directly block the replication o f viruses by inhibition of protein synthesis of viral polypeptides, viral R N A degradation, and editing of viral R N A 21 to inhibit replication, respectively. However, IFNs can upregulate hundreds of different gene products that can regulate anti-viral as well as anti-proliferative and immunomodulatory effects. IFNs can upregulate many cytokines and chemokines (such as interleukins, T N F - a , and R A N T E S ) which can recruit and activate both the innate and adaptive immune cells. In turn, IFN-y expression is upregulated by the activation of I L - l 2 and I L - l 8 family o f cytokines. IFN-a/p and IFN-y lead to increased levels of class I M H C molecules and the development o f CD8+ T-cell responses, whereas IFN-y is an efficient inducer o f M H C class II molecules in a variety of cell types including phagocytes, endothelial cells, epithelial cells, and CD4+ T-cells. Moreover, IFN-y has been known to modulate cell death by induction and/or inactivation of pro-apoptotic and pro-survival proteins. Our laboratory has shown that overexpression of IFN-y inducible GTPase (IGTP) results in activation of the PI3 -kinase/Akt survival pathway and inhibition of apoptosis in CVB3-infected cells [100]. A s well , the PI3-kinase pathway can mediate both apoptotic and survival activities in IFN-mediated signalling: IFN-a/p-activated PI3 -kinase signalling mediates survival signals through activation of A k t or P K C 8 , in a cell-type specific manner. In contrast, PI3-kinase activation of mammalian target o f rapamycin (mTOR) kinase is involved in apoptosis in other cell types (personal communication). Thus, IFN-mediated PI3-kinase signaling regulates cell death and survival dependent on the cell type and viral strain [98]. Extensive studies have shown the beneficial effects of interferon therapy in myocarditis by directly l imiting virus replication and dissemination, leading to an improvement in disease prognosis. I F N - a , IFN-P, and IFN-y treatments in myocarditic mice and humans resulted in reduced myocardial virus titers, inflammatory infiltration, and myocardial damage [103,104]. The effects of I F N - a was confirmed in vitro as evidenced by a reduction in virus replication 22 and cell death following administration of this molecule to cultured human pediatric fibroblasts infected with coxsackievirus [105]. In CVB3-infected mice lacking the IFN-p gene, an increase in susceptibility to infection, decrease in survival, and cardiomyocyte deterioration and disruption were observed [ 106]. The effect of IFN-y treatment to virus-infected mice was similar. Expression of recombinant IFN-y protected CVB3-infected mice by decreasing the viral load and spread as well as tissue destruction [107]. In IFN-y deficient mice infected with C V B 3 , extensive fibrosis, increased numbers of activated mast cells with concurrent upregulation of cytokines (TGF-(3, I L - l b , and IL-4), increased histamine levels, and reduced survival was observed [81,91]. Despite similar outcomes between the different I F N treatments, the antiviral mechanisms appear to be different. In CVB3-infected human myocardial fibroblasts treated with IFN-a , IFN-P, or IFN-y, differences in cytokine induction were observed [108]. The cytokine induction activity of IFN-P is more similar to IFN-y than I F N - a despite belonging to the same class, as evidenced by the reduction o f IL-6 and IL-8 expression by both IFN-P and IFN-y, while I F N - a had no such effect. However, ablation o f the common receptor for I F N - a and IFN-p in CVB3-infected mice resulted in early mortality of 100% of the infected mice while death of IFN-y receptor knockout (KO) mice was rare [109]. The exact mechanisms by which each interferon operates remain to be elucidated. Whi le single I F N gene deficiencies in mice lead to increased susceptibility to viral infections, double knockouts o f IFN-y and IFNa /p in mice are extremely sensitive to viral infections due to a reduction in both the innate and adaptive immunity, leading to persistence o f virus and protraction o f inflammation [110,111]. This highlights the importance of both the innate and adaptive immunity in anti-viral response. 23 1.5.2.2 Adaptive Immunity The role of the adaptive immune system, which consists of B- and T-lymphocytes, in CVB3 virus is still highly debated, in particular the roles of CD4+ and CD8+ T-cells. Lymphocytes are first detected in the myocardium from 4-5 days pi in the CVB3 mouse model and T-cells constitute the largest immune subset in the myocardium of mice and patients presenting with myocarditis, although B-cells are also minimally detected [49,112-114]. However, the importance of B-cells in CVB3-induced myocarditis is apparent as evidenced in the experiment by Mena et al [115]. This group showed that in B-cell KO mice following coxsackievirus infection, the viral load was considerably lower but histological outcome, inflammation, and mortality were more severe than controls during the acute phase. However, during the chronic phase, increased scarring and dilatation were observed in the B-cell KO mice in association with a high number of viral particles which were still detected within the myocardium, whereas the control mice suffered significantly less morbidity, underlining the importance of humoral immunity in the control and eradication of this virus. However, some studies suggest that the anti-virus effects of B-cells are offset by the production of auto-antibodies against host heart antigens, which may lead to autoimmune myocarditis [116-118]. Potentially autoreactive antibodies against cardiac myosin, actin, and other heart-specific antigens have been shown in myocarditic patients as well as mice [117-119]. Adoptive transfer of B-cells from myocarditis patients into uninfected SCID mice was shown to result in disease in these mice, suggesting a link between autoreactive antibodies and the initiation of myocarditis [120]. It has been suggested that these autoantibodies can cause autoimmune myocarditis via: 1) T-cell mediated cytolysis of uninfected cardiomyocytes through recognition of self-antigens or 2) viral mimicry, whereby viral antigens that closely resemble self proteins 24 induce activation of antibodies capable of recognizing self antigens, such that chronic inflammation and extensive loss of viable myocardium occur [116,121,122]. Thus far, only circumstantial evidence supports autoimmunity myocarditis via autoantibodies and more extensive studies are needed to support a definitive link. Many mouse models have been developed to study the roles of T-cells in myocarditis, mostly utilizing genetically modified mice that lack various T-cell specific components. CVB3 infection of SCID mice, which lack both T-cells and B-cells, produced a more severe histopathological disease than control mice [48,120]. These studies show that direct virus-induced cytolysis increases cardiac injury and host immunity is protective during the early phase of infection. However, despite extensive research utilizing genetically altered animals, there is still no general consensus on the roles of T-cells in the later stages of myocarditis. Kishimoto et al. [123] infected T cell-deficient, athymic nude mice with CVB3 and showed that acutely, myocardial virus titer, histopathological features, and anti-virus neutralizing antibodies were similar in both nude mice and control groups but progressive severe cardiac injury was apparent only in the control mice. Further, Henke et al. [61] observed similar histological results after depletion of CD8+ T-cells from CVB3-infected CD4+ K O mice but additionally, they showed that viral load was also decreased in the T-cell depleted mice, suggesting that lack of both CD4+ and CD8+ T-cells decreases virus replication, inflammation, and cardiac injury. This is in accordance to the studies performed by Opavsky and colleagues, who infected mice deficient in CD4+, CD8+, both CD4+ and CD8+, and T-cell receptor (TCR, which lack the a/p T-cell subtype) with CVB3. They observed that the CD4+/CD8+ double K O and TCR K O mice suffered less severe cardiac damage, as evidenced by reductions in inflammatory infiltrate and necrosis, and mortality than wildtype (WT) controls [124]. 25 However, these mice exhibited comparable amounts of neutralizing ant i -CVB antibodies and myocardial virus titers between K O and W T mice. Therefore, despite utilizing different mouse models to produce T-lymphocyte deficiency, there is general agreement that during the chronic stage o f myocarditis, lack of T-cells improve the outcome of infected mice, with or without a decrease in viral load. Opavsky et al. further dissected the roles of CD4+ and CD8+ T-cells in single K O experiments. Following C V B 3 infection, CD4+ T-cell K O mice acutely showed less inflammation and necrosis than W T , while the histopathological features were similar during the chronic stage. This contrasts the experiments performed by Henke's group who also infected C D 4 K O mice, albeit of a different background strain, and showed that during the acute phase, the C D 4 K O mice experienced a reduction in inflammation and cardiac injury as well as higher resistance to the virus as compared to W T [61]. However, the infected C D 4 K O mice developed severe myocarditis and suffered more extensive cardiac injury during the chronic stage. Leipner et al. further confirmed these results by infecting M H C II K O mice, which also lack CD4+ molecules, and observed less severe acute myocarditis but protraction o f inflammation, severe fibrosis, virus persistence, and a weak IgG response, with absence of virus neutralizing antibodies [125]. Further contradictory results are observed in CD8+ T-cell deficient mice following C V B 3 infection. Opavsky et al. showed that infected C D 8 K O mice exhibited a similar degree of inflammation and necrosis as controls during the acute phase, while chronically they experienced less cardiac necrosis but higher mortality. Kl ingel and colleagues infected mice deficient in P-macroglobulin, which also lack CD8+ T-cells, and observed an increase in viral titers and acute inflammation, a decrease in IFN-y secretion, and delayed production of 26 neutralizing Abs, which may all contribute to the development of chronic myocarditis seen in these mice [126]. This group also infected mice lacking perforin, which is the major cytotoxic effector o f CD8+ cytolytic T-cells, thus reducing their activity, and in contrast to the (3-macroglobulin K O mice, the histopathological features were similar to W T mice, suggesting that CD8+ T-cells are important in viral clearance and amelioration o f chronic myocarditis independent o f perforin activity. These results contradicts those of Henke and colleagues, who also infected P-macroglobulin K O mice with C V B 3 and observed an amelioration o f myocarditis, as evidenced by lower mortality, viral load, and inflammation [61]. Despite using genetically modified mice from the same background strain, these two authors obtained contradicting results, which highlights the need to perform more exhaustive experiments to dissect out the role of these cell types. Table 1.4 summarizes the results of T-cell deficiency in viral myocarditis from the different research groups. 27 Table 1.4: Comparison of results from T-cell deficient mice in viral myocarditis. The following studies used transgenic mice deficient in various immune factors to investigate the importance of T-cells in myocarditis. In general, impairment of T-cell function decreased inflammation and cardiac injury but ablation of specific subtypes of T-cells gave contrasting results. Genotypes Back-ground Strain Phenorypes Exhibited During Myocarditis Research Group SCID: Lack both T- and B-cells Acute myocarditis was very severe due to uncontrolled viral replication & dissemination, leading to increased myocytolysis. Chow et al., 1992 Nude: Lack all T-cells Balb/c Similar disease severity as WT during acute phase. Less inflammation & injury during chronic phase. Kishimoto et al., 1990 ra/B TCR KO Mice: Lack all T-cells with a/B TCR A/J Decreased inflammation, necrosis, and mortality as compared to WT, with no associated increase in viral titer. Opavsky et al., 1999 CD4/CD8 Double KO: Lack all CD4+ and CD8+ T-cells A/J Similar to a/B TCR KO with decreased inflammation, necrosis, and mortality as compared to WT, with no associated increase in viral titer. Opavsky et al., 1999 CD8+ T-cell Depletion in CD4KO Mice: Lack all CD4+ and CD8+ T-cells C57/B16 Decreased acute myocarditis as evidenced by reduction in inflammation, necrotic injury, and mortality, despite an increase in viral load. Henke et al., 1995 CD4+ KO Mice A/J Acutely experienced less inflammation and necrosis than WT, but disease progression was similar during chronic phase Opavsky et al., 1999 MHCII KO: No CD4+ T-cells C57/B16 Delayed inflammation and less injury during acute phase. Persistence of immune cells & virus, increased fibrosis, and absense of neutralizing virus antibodies. Leipner et al., 1999 CD4+ KO Mice C57/B16 Acutely exhibited less inflammation, injury, and susceptibility to virus but developed more severe disease during chronic phase. Henke et al., 1995 CD8+ KO Mice A/J Acutely exhibited similar degree of inflammation & injury as WT, but suffered more deaths despite less cytolysis during chronic phase. Opavsky et al., 1999 B2-Microglobulin KO Mice: Lack CD8+ T-cells C57/B16 Prevention of myocarditic disease as evidenced by lack of inflammation and cardiac injury with decreased viral load and mortality. Henke et al., 1995 B2-Microglobulin KO Mice: Lack CD8+ T-cells C57/B16 Acutely & chronically worse than WT, as shown by increased virus persistence and inflammation, with reductions in IFN-y and neutralizing Abs Klingel et al., 2003 28 Table 1.5: TNF family members: co-stimulatory molecules consisting of receptor/ligand pair. In general, the receptors are found on T-cells and the ligands are located on immune cells as well as activated somatic cells. It is mandatory for T-cells to receive two signals for proper activation: one for the T-cell receptor and the other from attachment of the co-stimulatory receptor to its ligand. R E C E P T O R S LIGANDS 4 - I B B 4 - 1 B B L CD27 CD70 CD30 C D 3 0 L O X 4 0 O X 4 0 L H V E M L I G H T G I T R G I T R L In an effort to investigate the importance of T-lymphocytes in myocarditis, recent studies have focused on the co-stimulatory molecules. Activation of T-cells requires two signals: the first signal from the T C R interaction with M H C class molecules presenting antigen peptides and the second signal is generated by co-stimulatory molecules. This second signal is necessary in order to prevent the T-cell from entering an anergic state and to enhance activation stemming from the T C R molecules as well as to promote cell proliferation, regulate cell survival, and induce effector functions [127]. The expression of the co-stimulatory molecules can be co-dependent and quantitative, resulting in enhanced signals, or qualitative, in such that the different co-stimulatory molecules activate distinct pathways. Generally, the co-stimulatory receptor molecule is expressed by T-cells and their ligand is expressed by antigen-presenting cells, other immune cell types, as well as on injured mesenchymal cells [127]. There is low level constitutive expression of some co-stimulatory receptors on naive T-cells but expression o f these molecules is usually induced upon stimulation, such as virus infection, and is dependent on various factors, including the stage of T-cell development, state 29 of the immune response, causative agent of inflammation (type o f virus and dose), and activation status of other immune cells [127,128]. There are two families of co-stimulatory molecules: 1) the immunoglobulin and 2) the tumour necrosis factor receptor (TNFR) superfamilies. The most common receptor/ligand pair of the immunoglobulin co-stimulatory family is CD28 receptor and its ligand B7 , which are widely considered to be the primary co-stimulatory complex for initial expansion and survival o f T cells [129]. Emerging evidence has shown that the T N F R family of molecules also plays vital roles in stimulation of T-lymphocyte during viral infections. The T N F R family consists of many receptor/ligand pairs, as shown in Table 1.5. Seko et al. performed extensive studies in C V B 3-infected mice to investigate the roles of the various members of the T N F R superfamily i n myocarditis [130-132]. This group investigated the expression levels o f 4-1 B B L , O X 4 0 L , C D 3 0 L , CD40, and C D 2 7 L in human and mouse hearts with D C M and/or myocarditis. Their primary finding was that inhibition o f interaction between the 4 - I B B receptor and its ligand, by treatment with neutralizing an t i -4 - lBBL antibodies, greatly ameliorated inflammation and decreased amount and size of lesions in CVB3-infected mice, whereas inhibition of the other pathways had little or minimal effects. This suggests that the 4-1BB pathway, but not the other T N F R pathways, may play a vital role in viral myocarditis. The 4 - I B B molecule is a cell membrane protein receptor, which is usually but not exclusively induced on activated T-cells, and act to influence effector functions and proliferation in the late stage of the immune response. This protein pathway may act in concert to stimulate T-cells with the CD28/B7 pathway or may act in an independent fashion [127]. The most characterized ligand for this protein is 4 - 1 B B L , which is constitutively expressed on plasma monocytes and dendritic cells but can also be induced on B-cells, activated 30 macrophages, as well as cardiomyocytes [133,134]. CD40 /CD40L ligation, lipopolysaccharide (LPS), as well as other toxic and infectious insults can stimulate 4 - 1 B B L expression [140]. Upon activation, 4 - I B B ligand-expressing cells are documented to upregulate the secretion of cytokines, such as IL-12 and IFN-y [136]. Signalling through 4 - I B B plays a vital role in: prevention of activation-induced cell death, promotion of cardiac allograft and skin transplant rejections, enhancement of integrin-mediated cell adhesion, eradication of established tumours, and upregulation of T-cell cytolytic potential, as well as induction o f anti-viral defenses [137-142]. Many studies have recently manipulated the 4 -1BBL/4 -1BB pathway to abate the immune response in many inflammatory diseases, such as cancer and rheumatoid arthritis. In cancer, tumours have altered antigens which may be recognized by subsets o f CD8+ T-cells. Co-stimulation by the 4 - 1 B B L / 4 - 1 B B pathway can enhance CD8+ T-cell proliferation and destruction o f the tumour cells. Studies have characterized the expression o f 4 - 1 B B L on cancer cells and signalling from this ligand induces upregulation o f cytokines, such as IL-8 [136]. Interactions between 4-1BB on activated T-cells and 4 - 1 B B L on the tumours also increase the secretion of INF-y, thus enhancing the immune response and facilitating tumour clearance. However, tumours may counteract this protective mechanism and escape immune surveillance through cleavage of 4 - 1 B B L from tumour cells, producing soluble 4 - 1 B B L , which is MMP-dependent [143]. In rheumatoid arthritis, investigators believe that this pathway plays a role in T-cell mediated humoral immune response and the production o f autoantibodies. Mittler et al. suggested that antibodies against 4 - I B B given to animals within one week of foreign antigen immunization abrogated the immune response through induction o f T-helper cell anergy 31 [144]. In experimental collagen-induced arthritis, a mouse model of rheumatoid arthritis, researchers have shown that agonistic ant i -4- lBB treatment, given immediately after immunization with collagen type II or when disease has already set in, abrogates disease and confers longterm protection [145]. Investigators believe that in this model, the agonist anti-4 - I B B antibodies upregulate a subset of INF-y-producing CD8+ T-cells that decrease CD4+ T-cells thus also decreasing the production of autoantibodies. The 4 - 1 B B L plays a crucial role in many infectious diseases, such as those due to infection with Lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), influenza virus vaccinia, herpes simplex 1 and 2, and the murine 7 herpes virus, as well as in several bacterial infections [146,147]. However, the mechanisms by which this pathway functions in microbial immunity is still unclear. Infection of 4 -1BB- and 4- lBBL-defic ient transgenic mice with LCMV or influenza virus showed negligible effects during the primary immune response and viral infection, but upon restimulation with the antigen, there was a major defect in the recall response, suggesting that the 4 - 1 B B L / 4 - 1 B B pathway plays a vital role in secondary T-cell responses [137,148]. In herpes stroma keratitis infection of the eye, depletion of 4 - I B B and 4 - 1 B B L using knockout mice and antagonistic antibodies, respectively, greatly inhibited disease severity via downregulation of T-cell recruitment into the affected area [149]. These studies all report lower frequencies of antigen-reactive effector T-cells only during the late immune response, which leads to fewer memory T-cells, possibly due to induction of extensive cell death since 4 - I B B provides survival signals by upregulating B C L -X L and B F L 1 to activated T-lymphocytes [142]. Despite these promising studies into the role of the 4 - I B B pathway in viral diseases and myocarditis, the mechanisms by which it functions are still largely unclear. It has been 32 shown that both 4 - I B B receptor and its ligand are upregulated in cardiomyocytes under stressful conditions, such as C V B 3 infection, cardiotoxicity, and hypoxia (personal communication, Amgen) [131]. Unpublished data (Amgen) suggests that stimulation of 4-1BB receptor on cardiomyocytes may not only activate inflammatory cells, but may also induce apoptosis involving mitochondrial disruption. Studies by Seko et al. found increased expression o f 4 - I B B ligand in myocarditic and dilated cardiomyopathic myocardium in mouse and human [131,132]. This group also found that administration o f a monoclonal antibody to 4 - 1 B B L , which inhibited its function, to virus-infected mice greatly reduced inflammation but increased cytokine production in isolated cardiomyocytes. Agonist antibodies against 4 - I B B greatly caused deterioration in adriamycin-induced D C M in a mouse model but antagonist studies completely protected the injured animals. Further, in genetically modified mice deficient in 4 - 1 B B L treated with adriamycin, cardiac injury was completely inhibited (personal communication, Amgen). Thus, the role of the 4 - I B B pathway may be more complex and requires further investigations. 1.6 Reparation and Reclamation 1.6.1 Injury and Wound Healing in the Heart The major outcome of myocarditis regardless of etiology is loss of viable myocardium and a decrease in ventricular function. It has been suggested that the reparation process may be similar among different types of cardiac disorders, irrespective of initial cause of injury [150]. Following an injurious attack to the heart, both the normal and injured regions of the myocardium are subjected to biological processes, which result in cardiomyocyte hypertrophy and cellular alterations, reactive and reparative fibrosis, and matrix expansion, ultimately 33 leading to enlargement and alterations to the structure of the left ventricle [150]. The exact mechanisms, by which complete reparation and resolution of cardiac damage occur, are still elusive. The ability of the host to completely clear the virus early on in the infection w i l l greatly lessen the chance of longterm damage. The result of viral clearance is inevitably cytolysis but the extent of myocardial damage is dependent on many factors, such as gender, genetic composition, immune status, initial amount of infecting virus, virus strain, previous infections, and persistence of virus. Wound healing begins immediately following cardiac injury, whereby infected cardiomyocytes release soluble signalling molecules, including cytokines, chemokines and neurohormones, to activate the innate immune system to eliminate the virus and recruit fibroblasts to instigate the reparation process, as depicted in Figure 1.5. 34 Figure 1.5: A simplified schematic representation of the wound healing process. Cellular injury activates intracellular signaling transduction pathways that lead to secretion of cytokines and growth factors, such as TGF-|3 and interferons, which recruit fibroblasts to initiate the reparation process and immune cells to eliminate infectious agents and damaged cells. Granulation tissue is laid down by the fibroblasts to replace the dead cells, in order to maintain tissue structure. Proteases, such as M M P s , are secreted by infiltrating as well as resident cells to remodel the granulation tissue, a continuous process that can last months to years. Maladaptive remodeling may lead to inadequate maintenance of tissue structure and function, thus resulting in organ failure. T G F - p TNF-oc interleukins Injury Cytokine release Activation & recruitment of fibroblasts chemokines interleukins nterferons Immune cell infiltration Cell proliferation & replacement fibrosis granulocytes macrophages T-lymphocytes TGF -P 1 collagens Extracellular remodeling proteases (MMPs,cathepsins) Heart failure Maintain structure & function The recruitment and activation of innate immune cells lead to phagocytosis of infected cells in a non-specific fashion and the injured cells secrete anti-viral IFNs to further combat the infection, as described above. Even though P M N s are usually associated with bacterial infections, they are the first immune cell type to enter the wound site because they secrete a variety of cytokines, growth factors, and proteases to pave way for the next wave o f 3 5 immune cells [58,151,152]. Cytolysis due to virus dissemination is usually a necrotic event, which results in recruitment of P M N s to clear cellular and matrix debris and in further secretion o f signalling molecules to propagate the immune response. P M N s also secrete a large amount of proteases, such as neutrophil elastases and M M P s , which degrade the matrix to further modulate migration of incoming cells but may also lead to loss o f proper matrix organization. A s shown by Kishimoto et al., during the early phase of myocarditis, cardiac cell injury and death result in myocyte slippage due to disruption and disorganization o f the fine reticulin fibers, consisting of mostly collagen type III fibers, and loss of extracellular connections between adjacent cardiomyocytes [153]. Tissue damage also leads to extravasation of plasma fibrin and fibronectin into the myocardium to form a provisional matrix or granulation tissue, which supports the migration and proliferation of infiltrating inflammatory cells, myofibroblasts, and endothelial cells to develop an extensive microvascular network [154-157]. The second wave o f immune cells consists o f monocyte-derived macrophages and in early studies of wound healing, Leibovich showed that macrophages were pinnacle in wound healing [158]. In this study, administration of both hydrocortisone, which elicited monocytopenia, and subcutaneous anti-macrophage serum to skin wounds in mice resulted in almost complete absence of macrophages. Fibrin levels were elevated, and clearance of fibrin, neutrophils, and other cellular and matrix debris from these wounds was delayed. The initial provisional matrix is degraded by proteolytic enzymes and impairment of this clearance results in delayed granulation tissue formation and defective healing [159]. This and other studies highlight the importance o f the innate immunity in initiation of the wound healing process [160,161]. Leibovich et al. also showed a delay in the recruitment and activation of myofibroblasts. After the breakdown o f the initial 36 provisional matrix, myofibroblasts play the most important role in wound healing because they are primarily responsible for massive E C M synthesis and deposition, without which mature scars cannot develop and wound healing is inhibited. The origin o f myofibroblasts is still unclear but previous studies have suggested that fibroblasts can be stimulated by various cytokines, such as TGF- f i , to differentiate into myofibroblasts, o f which the distinguishing features include the expression of a-smooth muscle actin and the ability to contract [157]. Myofibroblasts are found abundantly in areas undergoing extensive remodeling and they can persist throughout healing and beyond, having been found in human M I scars decades after the initial injury [162]. They secrete abundant amount of matrix, usually consisting of hyaluronan, tenascin, cellular fibronectin, and predominately collagen [157]. Hyaluronan is a glycosaminoglycan that is extremely hydrophilic and though its function in myocarditis is unknown, it may function to regulate cell adhesion and division. Tenascin is a fetal protein found to re-express after myocardial injury and may modulate the attachment o f cardiomyocytes to matrix, enhance recruitment and differentiation of myofibroblasts, and stimulate synthesis of proteases [162]. Fibronectin accumulation is especially important in wound healing due to its regulation of fibrillar collagen synthesis and organization, and was found to be crucial in regulation of interstitial matrix and normal reticulin fiber formation during CVB3-induced myocarditis in mice [156,164]. The provisional matrix then evolves into a collagenous lesion via abundant production and secretion o f collagen type III by myofibroblasts, followed by collagen type I accumulation. Collagen type I forms thicker and more tensile fibers as compared to collagen type III fibers, which are thinner and have increased elasticity [165]. In myocarditis, the transition to a collagen matrix is visualized as mi ld fibrosis stained with picrosirius red at approximately 14 days post-infection (pi) but 37 extensive disruption and collapse of reticulin fibers can still be detected in the necrotic regions [153,166]. Myofibroblasts not only counteract the loss of viable myocardium through synthesis o f matrix but they also form highly organized arrays and along with their contractile properties, the provisional matrix not only has tensile strength but also aids in maintenance o f ventricular wall contraction during wound healing [162]. This process is usually regulated by pro-fibrotic molecules, such as TGF-J3, connective tissue growth factor, and proteases ( M M P and cathepsins). Accumulating evidence has suggested that neurohormonal factors such as angiotensin II (Ang II), aldosterone, endothelin-1 (ET-1) and norepinephrine are important modulators of the wound healing process. A l l o f these factors induce hypertrophy of cardiomyocytes and increase myofibroblasts proliferation and collagen synthesis [167]. The rapid synthesis, secretion, and accumulation of collagen result in immature granulation tissue characterized by improper organization, array, and orientation of the fibers at the injury site. Subsequently, the lesion matures, involving resorption of the provisional matrix mediated by proteases, collagen type I accumulation, and development o f intermolecular cross links in collagen fibers to increase resistance to distension and tensile strength [168]. This maturation process also involves a clearance of cells in the provisional matrix, which has been reported to be mediated by apoptosis, essentially forming an acellular and avascular fibrotic scar [162]. This process is observed at approximately 60 days pi in CVB3-infected mice, when multiple densely fibrotic and calcified scars with paucity o f cells are detected at the injury sites. The reticulin network in these mice appears thin at the injury-sites but is thickened in normal heart regions [153,166]. Therefore, not only are the infected wound sites affected but remote areas of the heart also undergo remodeling. Fibrosis leads to ineffective contraction and improper electrical signal conduction while degradation of the 38 interstitial collagen network may result in the loss of structural support which can lead to wall thinning and L V dilatation. Remodeling of the heart is a continuous process and is necessary to maintain proper cardiac structure and function. It has been postulated that the inflammatory response is primarily responsible for continual remodeling [58,153]. Using different variants o f virus that can elicit varying degrees of inflammation, Leslie et al. showed that persistent immune infiltration o f the myocardium following C V B 3 infection caused an increase in fibrosis and thinning o f the reticulin fibers in scar tissue while thickening of the reticulin network in areas remote from scars was observed [166]. Only the virus variants associated with chronic inflammation developed interstitial fibrosis and abnormal reticulin alterations. Thus, immune cells are like a double-edged sword: they are the direct effectors of viral clearance and myofibroblast recruitment and activation, but they can also distort the fibrotic and remodeling processes by secreting a variety of signalling molecules, such as cytokines, chemokines, growth factors, and proteases. 1.6.2 Proteases and Matrix Remodeling During wound healing the deposition and resorption o f matrix is crucial to the maturation of the scar lesions because this rearrangement enables migration o f infiltrates into the tissue as well as reorganization of myofibroblasts and cardiac cells within the wound healing area [169]. There are only a limited number of proteases that have matrix degrading capabilities, including cathepsins, a few serine proteinases such as plasmin, and M M P s , and they likely work in concert with each other [167,170]. 39 1.6.2.1 Cathepsins Cathepsins are cysteine proteases, which are usually localized to the cell membrane or in intracellular endolysosomal compartments to mediate protein turnover and degrade collagen under acidic conditions [171]. Our laboratory observed an increase in the transcriptional expression of most cathepsins during myocarditis, using an Affymetrix microarray platform (Figure 1.6). Upon certain stimulation, cathepsins, in particular types B , L , and S, may be released into the extracellular compartment to cleave E C M proteins such as laminin, type IV collagen of the basal lamina, tenascin, and elastin as well as intracellular degradation o f collagens [171,172]. Cathepsin B is the most widely studied cysteine protease and it has been suggested to increase inflammation by mediating migration through their activity at the cell surface of migrating cells [173]. This enzyme has both intracellular and extracellular functions, including type IV collagenolytic capabilities and proteolysis o f laminin, which involves the plasminogen/plasmin system [172,174]. Further, previous studies have shown increased cathepsin B in D C M patients and post-MI canines and it has been suggested that this enzyme is a prominent protease involved in cell death as well as degradation of myofibrillar proteins, in particular myosin heavy chain, a-actinin and troponin-I [175,176]. Cathepsins L and S are also capable o f intracellular and extracellular collagenolytic activities and recent investigations have shown that these two enzymes are also potent elastases, whereby they are almost as active as pancreatic and neutrophil elastases (both serine proteases) and M M P s . In particular, cathepsin knockout mice had progressive cardiac dysfunction culminating to D C M without any external stimuli, thus confirming the importance o f this protease in maintenance of cardiac structure and remodeling [177]. However, CVB3-infect ion of cathepsin L knockout mice did not exhibit any difference in 40 cardiac viral titers and histopathology as compared to infected W T mice during acute myocarditis, which suggests that this enzyme may function during a later stage (Dr. Brad Spiller, personal communication). Using cathepsin S knockout cells and mice, investigators have shown that endothelial cells lacking this enzyme have impaired ability to degrade elastin and type I V collagen as well as markedly reduced migration, suggesting that cathepsin may play an important role in angiogenesis and vascular remodeling [178]. Cathepsins have been shown to be expressed by cardiomyocytes, smooth muscle cells, fibroblasts, as well as macrophages and T-cells, all o f which are found within the myocarditic heart, and their production and activity are regulated by T N F - a , T G F - p , and I L - l p [170,172,179]. However, the roles o f cathepsins in myocardial diseases and ventricular remodeling are still largely unknown due to paucity of research in this area. Figure 1.6: Affymetrix oligonucleotide microarray analysis of cathepsin expression in CVB3-infected mouse hearts (Reprinted with permission from Dr. Bobby Yanagawa). A l l cathepsins are highly upregulated following C V B 3 infection in mice. On average, transcriptional expression of cathepsins is increased by more than 5 fold as compared to sham. Days Post-Infection S-> o WD a « -e U 2 "© to C t s f C t s e 41 Figure 1.7: The plasminogen system and its role in remodeling (Adapted from Collen et al. [180]). Plasminogen can be cleaved into active plasmin by either tPA or u P A , the activities of which are inhibited to PAIs. Plasmin is known to be a major activator of M M P s . Plasminogen t-PA PAI -H-PAI u-PA:u-PAR Plasmin a 2-Antiplasmin Fibrin • F D P Pro-MMP- MMP A I TIMP-ECM Degradation 1.6.2.2 Plasminogen System The plasminogen and M M P systems are more extensively studied in the realm o f cardiac diseases and their functions are frequently interconnected. Figure 1.7 shows a simplified scheme of the plasminogen system, which consists o f the tissue-type plasminogen activator (tPA), urokinase-type P A (uPA), the plasminogen activator inhibitor (PAI-1) which inhibits both P A s , the precursor plasminogen, the active enzyme plasmin, and serine protease inhibitors (serpins) which block plasmin activity [180]. Briefly, induction of tPA or u P A expression in the tissue leads to cleavage o f precursor plasminogen, which extravasates from 42 circulation into the myocardium, into active plasmin. PAI-1 is the major inhibitor of the P A enzymes and serpins inhibit the activity of plasmin, which is a major effector of matrix remodeling. The use of genetically modified mice has provided some evidence in the importance o f the plasminogen system in cardiac remodeling. Using mice lacking plasminogen in an M I model, it was shown that these mice are protected against ventricular wall rupture, but fail to heal the ischemic myocardium, which remains largely devoid o f fibrous healing, leukocytes, endothelial cells, and fibroblasts [159]. Further, mice deficient in PAI -1 , which essentially overexpress both P A s and therefore increase plasmin, develop cardiac macrophage accumulation, and fibrosis following M I [181]. This suggests that plasmin plays an important role in cardiac wound healing but the relative importance of its activators is unknown because despite a common substrate, the P A enzymes have entirely different function, are cell-specific, and temporally modulated. The function of tPA in cardiac remodeling lies predominately in fibrinolysis. This enzyme is expressed by fibroblasts early in wound healing and tPA is still increased as healing proceeds [182]. Fibrinogen-deficient mice were shown to have decreased infarct size and inflammation following M I and in skin wound healing models, these fibrinogen K O mice experienced decreased tensile lesion strength and delayed wound closure, albeit injury resolution was not different between K O and W T [183-185]. This was confirmed in a skin wounding model of tPA K O mice, where reduced fibrinolysis and fibrin persistence were associated with an enhanced accumulation of collagen and the development of skin fibrosis [186]. Based on these results, tPA and its effect on the fibrin provisional matrix may regulate cellular migration, fibrosis, and rate of healing, but their longterm effects in cardiac repair is still unclear, as evidenced by the absence of active tPA in mature scars and tPA K O mice 43 experienced similar degrees of fibrosis, dilatation, and cardiac dysfunction as W T mice in pressure overload-induced hypertrophy and chronic cardiac hypoxia models [187-189]. The u P A protein on the other hand has been shown to have more direct effects on remodeling. Human and animal studies suggest that increased u P A activity contribute to the pathogenesis of cardiac fibrosis. Mice deficient in u P A have been studied in the context of pressure overload-induced hypertrophy, pulmonary hypertension, and coronary disease. In both hypertension models, u P A K O mice exhibited minimal cardiomyocyte hypertrophy, myocytolysis, fibrosis, interstitial collagen network degradation, and cardiomyocyte slippage [188,189]. Consequently, L V contractility was enhanced and normal fractional shortening was preserved without signs o f cardiac failure or pulmonary congestion in the K O mice [188]. However, in the M I model, despite complete protection against cardiac rupture in u P A K O mice, the animals experienced impairment in scar formation, revascularization, and died o f cardiac failure due to depressed contractility, arrhythmias, and ischemia [190]. This phenomenon was attributed to lack of infiltration of inflammatory cells, in particular macrophages, and fibroblasts, as both cell types require u P A to migrate into the damaged myocardium. It has been shown that u P A is secreted by infiltrating immune and fibroblastic cells, thus this enzyme is necessary for cellular migration. There are two mechanisms by which u P A can mediate cardiac repair: 1) induction of matrix production through myofibroblast activation and 2) degradation of E C M via either direct proteolysis of the matrix or activation of other proteases, such as M M P s . The u P A enyzme can also cleave precursor forms of growth factors, such as TGF-p \ to generate active molecules thus activating myofibroblasts to increase matrix production [188]. Further, plasmin activation of myofibroblasts may result in the production of various cytokines and growth factors, such as 44 interleukins and T G F - p , which can also induce synthesis and activation o f M M P s [191,192]. The plasminogen system can also mediate cardiac repair and remodeling through activation and regulation of M M P s . Plasmin is a potent activator of precursor M M P s , via proteolytic cleavage of the latency-conferring propeptide of M M P s , and many studies have shown a synergistic relationship between them [193-195]. 1.6.2.3 Matrix Metalloproteinases ( M M P s ) M M P s are calcium- and zinc-dependent enzymes that belong to the metzincin group o f proteases [196]. Currently, there are over 25 members in this family, as shown in Table 1.6, consisting of secreted free-form enzymes and cell surface-expressed M M P s . The archetypal structure o f M M P s is shown in Figure 1.8: 1) N-terminal signal peptide for extracellular secretion; 2) propeptide that confers latency by binding to the 3) catalytic region, which harbours a zinc ion that is crucial for activity, often followed by a 4) hemopexin domain that confers substrate specificity, and in the cell-membrane bound M M P s , there is a 5) transmembrane region [196]. 45 Figure 1.8: Archetypal structures of MMPs. A s shown in the left panel, M M P s are usually secreted as inactive zymogens with an N-terminal propeptide domain that blocks the catalytic domain by the cysteine-switch mechanism, where the cysteine in the propetide and three histidines in the catalytic domain coordinate with a zinc ion that is required for enyzymatic activity. Proteolytic cleavage of the propeptide releases the active enzyme. On the right is shown a membrane-type M M P structure, which is similar to the secreted form except for the addition of a transmembrane domain and a short cytoplasmic tail. The M T -M M P s are usually activated intracellularly by furin. Archetypal MMP Structure: Membrane-type MMP Structure: N-terminus Propeptide Domain Cys Zn Catalyti '*• Hemopexin —C-terminus N-terminus Propeptide Domain Cys JUT YM jflHUfl Catalytic "•* Hemopexin —C-terminus Cell Membrane Cytoplasmic Tail 46 Table 1.6: Matrix metalloproteinase family and known substrates. Currently, there are 23 known M M P s in this family of protease. Substrates include matrix components as well as other proteases, enzyme inhibitors, receptors, cytokines, and other bioactive molecules. Enzyme Other Names Molecular Weight (kDA) Collagen Substrates Additional Substrates MMP-1 (Interstitial, Fibroblast) Collagenase-1 51/45 I, II, III, VII, VIII, x Aggrecan, Gelatin, MMP-2, MMP-9 MMP-2 Type IV Collagenase, Gelatinase A 72/62 I, II, III, IV, V, VII, X, XI Aggrecan, Elastin, Fibronectin, Gelatin, MMP-9, MMP-13, Laminin MMP-3 Stromelysin-1 57/45 II, III, IV, IX, X, XI Aggrecan, Elastin, Fibronectin, Gelatin, MMP-8, MMP-13, MMP-7, Laminin, proTGF-b MMP-7 Matrilysin 28/19 IV, X Aggrecan, Elastin, Fibronectin, Gelatin, MMP-1, MMP-2, MMP-9, Laminin, proTNF, FAS ligand MMP-8 (Neutrophil) Collagenase-2 75/65 & 55/45 I, II, III, v, VII, VIII, X Aggrecan, Elastin, Fibronectin, Gelatin, Laminin MMP-9 Type IV Collagenase, Gelatinase B 92/82 IV, V, VII, X, XIV Aggrecan, Elastin, Fibronectin, Gelatin, proTGF-b, proIL-lb, proIL-8, proVEGF, Fibrin, Proteoglycans MMP-10 Stromelysin-2 57/44 III, IV, V Aggrecan, Elastin, Fibronectin, Gelatin, MMP-8, MMP-1, Laminin MMP-11 Stromelysin-3 51/44 Aggrecan, Fibronectin, Laminin MMP-12 (Macrophage) Metalloelastase 54/45/22 IV Elastin, Fibronectin, Gelatin, Plasmin, Laminin, proTNF MMP-13 Collagenase-3 60/48 I, II, III, IV Aggrecan, Gelatin MMP-14/MT1 -MMP 66/56 I, II, III Aggrecan, Elastin, Fibronectin, Gelatin, proMMP-2, proMMP-13, Laminin MMP-15/MT2-MMP 72/60 Fibronectin, Gelatin, MMP-2„Laminin MMP-16/MT3-MMP 64/52 MMP-2 MMP-17/MT4-MMP 57/53 Fibrin, Gelatin MMP-18 Xenopus Collagenase-4 70/53 MMP-19 54/45 IV Fibronectin, Aggrecan, COMP, Laminin, Gelatin MMP-20 Enamelysin 54/22 Aggrecan, Amelogenin, COMP MMP-23 CA-MMP 50/45 MMP-2 4/MT5-MMP 62 MMP-25/MT6-MMP Leukolysin 63/34/28 IV Gelatin, Fibronectin, Laminin, a-1-Proteinase Inhibitor MMP-26 Endometase, Matrilysin-2 28/19 IV MMP-27 59 MMP-2 8 Epilysin 62/58/50/46 47 M M P s are pleiotropic enzymes and are involved not only in matrix degradation but in many other crucial events, including cell migration, proliferation, apoptosis, morphogenesis, angiogenesis, and activation of other proteases [197,198]. A n imbalance in the activity o f M M P s has many detrimental effects, including dysregulation of the fibrotic process, deficiency in cellular migration, and improper wound healing. Therefore, due to these important functions, the regulation of M M P expression and activity is tightly controlled. Many of the M M P s are expressed only upon induction and the primary stimulators of M M P s are cytokines and growth hormones, such as interleukins (IL-l(3), IFNs, E G F , K G F , N G F , H G F , b F G F , V E G F , P D G F , TNF-oc, and TGF-(3 [199]. To further tighten regulation o f M M P expression, several cytokines and growth factors have been reported to regulate m R N A stability of M M P s [199,200]. TGF-p \ which is a major stimulator of fibrosis, not surprisingly inhibits the expression of most M M P s , except it has been reported to increase translation of M M P - 2 , M M P - 9 , and M M P - 1 3 by stabilizing their m R N A and preventing its degradation [199,201,202]. Upon translation, generally all M M P s , except the membrane-type M M P s , are directly secreted into the E C M , led by the signal peptide. The enzymes are secreted in a precursor latent form and require removal of the N-terminal prodomain, which confers latency to the enzyme by folding back and obstruction o f the catalytic domain [203-205]. A cysteine residue in the N-terminal end of M M P s interacts with Z n 2 + ions at the active site to neutralize all enzymatic activity. Disruption of this interaction, either via proteolytic cleavage of the prodomain (by proteases such as plasmin, other M M P s , or autocleavage) or modification of the cysteine-thiol group (reactive oxygen species), a process known as the cysteine-switch mechanism [206]. The membrane-type M M P s (MT1 to M T 6 - M M P s ) are also initially 48 synthesized as a precursor and they can either be processed extracellularly as the other M M P s , or they are proteolytically activated by intracellular enzymes, such as furin [206]. M M P s can diffuse through the E C M or remain in close apposition to the cell surface by binding to membrane-bound molecules, for example, M M P - 9 can bind to the hyaluronan receptor CD44 and M M P - 1 co-localizes with a 2 p i integrins in migrating cells, thus regulating pericellular and remote proteolysis [207,208]. Major endogenous inhibitors of M M P s are the tissue inhibitors o f metalloproteinases (TIMPs), which consists of 4 molecules, and each T I M P can inhibit the majority of M M P s though by varying kinetics [209]. Reports have also shown that M M P s can also be inhibited by several other peptides, such as tissue factor pathway inhibitor-2, a C-terminal fragment of the procollagen C-terminal proteinase enhancer protein, the secreted form of the large membrane-bound p-amyloid precursor protein, and a GPI-anchored glycoprotein R E C K (reversion-inducing-cysteine-rich protein with Kazal motifs), although the mechanisms and physiological significance are still largely unknown [210-213]. M M P s are also cleared from the system by binding to the plasma scavenger protein oc2-macroglobulin, a general endopeptidase inhibitor, and the whole complex attaches to the cell surface clearance receptor L R P (low density lipoprotein receptor-related protein), which triggers rapid internalization of the complex by receptor-mediated endocytosis [214]. Extensive studies on M M P expression and activation in myocardial diseases suggest that they are the most crucial family of proteases in cardiac reparation and remodeling [215,216]. The current paradigm in cardiac remodeling is that injury requires: 1) M M P -mediated cellular infiltration and E C M degradation during initiation o f cardiac repair and 2) a balance in E C M synthesis and MMP-mediated matrix degradation in order to prevent 49 cardiac fibrosis or the inverse from developing [151,169,217]. In general, inhibition or genetic deficiency of M M P s attenuates L V maladaptation to conditions such as pressure or volume overload-induced hypertrophy, M I , and tachycardia-induced heart failure, while more severe disease develops when M M P activities are enhanced [188,218-220]. Many studies have shown early upregulation of M M P s during cardiac injury, in particular M M P - 9 and M M P - 2 levels were increased within 1 day following M I in humans and mice [221]. Extensive studies using M M P deficient mice or administration of M M P inhibitors have shown improvement in cardiac function, structure, and overall mortality, which highlight the importance of M M P s in particular cardiac diseases. For example, ablation of M M P - 9 and overexpression of TIMP-1 via gene transfer attenuated L V remodeling and cardiac dysfunction using a pressure overload-induced hypertrophy model in mice, with TIMP-1 overexpression being more efficient since TIMP-1 can inhibit various M M P s other than M M P - 9 [188]. TIMP-3 ablation in mice mimicked D C M progression in humans, as evidenced by spontaneous L V dilatation, cardiomyocyte hypertrophy, contractile dysfunction, interstitial matrix disruption and reduction with elevated M M P - 9 activity, and activation of the cytokine system, in particular T N F - a [222,223]. This is not surprising since TIMP-3 has a large inhibition repertoire and can inhibit not only M M P s , but also T N F - a converting enzyme ( T A C E ) , which is the primary T N F - a activating molecule, thus directly affecting the cytokine profile. However, in studies using pharmacological inhibitors o f M M P s , success is more variable. Comparative studies of W T and TIMP-1 K O mice showed that TIMP-1 deficiency markedly exacerbated mortality, cardiac dysfunction, and interstitial matrix organization in mice following M I and administration of M M P inhibitor to these mice greatly alleviated the disease condition [224]. Administration of M M P inhibitors alone to 50 mice also greatly alleviated cardiac dysfunction and improved remodeling following ischemic injury but this has not been recapitulated in humans, as evidenced in the P R E M I E R (Prevention of Myocardial Infarction Early Remodeling) trial where patients were administered PG-116800, an oral M M P inhibitor, for 90 days following an M I attack and no significant improvement in L V diastolic/systolic volumes, ejection fraction, sphericity index, plus mortality or reinfarction were observed [225]. Further, it has been shown that in mice following M I , short term treatment with M M P inhibitor (up to 38 days post-MI) imparted a beneficial effect in regards to survival, cardiac function as measured using echocardiography, and interstitial matrix appearance while long term inhibition (over 3 months) had contrasting effects, as evidenced by an increase in mortality, cardiac hypertrophy, and interstitial fibrosis [226]. These studies highlight the importance of not only balancing the amount of M M P activity and E C M turnover but also the timing since M M P s have been shown to regulate many diverse biological processes beyond E C M proteolysis, including facilitation o f myofibroblast and immune cell migration, regulation of cytokines such as T N F - a and TGF-p , regulation o f cell death, cellular differentiation and proliferation, and vascularization [196]. Cardiac reparation and resolution is a highly complex process and the roles o f M M P s need to be further explored. 1.7 Sequelae The spontaneity and variability of myocarditis give rise to at least four possible outcomes, as depicted in Figure 1.9. The majority of myocarditis cases results in spontaneous and complete resolution of disease during the acute phase, despite the severity of symptoms, with minimal longterm cardiac injury, such that individuals with sub-clinical 51 symptoms and complete disease resolution often are ignorant o f contraction of disease. Fulminant myocarditis can result in acute D C M and C H F as a result of excessive necrosis and injury to the myocardium, leading to loss of viable muscle, structural damage such as thinning o f the ventricular walls, abnormal electrophysiology, and ventricular dysfunction [5]. This manifestation of myocarditis usually results in spontaneous resolution and is often j not acutely fatal. However, some forms of myocarditis, such as giant cell myocarditis, are frequently fulminant and may cause progressive heart failure or fatality [12,227]. Sub-acute and acute cases o f myocarditis may also result in D C M and C H F , despite mild primary clinical presentations. Extensive research suggests that a chronic sequela o f myocarditis is D C M , which may evolve months to years following the initial presentation [5,31,116]. In a subset o f cases, persistent infection by viral agents and/or autoimmunity has been postulated to result in chronic myocarditis, leading to D C M and C H F [47,49]. Virus genome, viral antigens, and anti-virus antibodies have been detected in chronic myocarditic and D C M patients using R T - P C R , ISH, immunological, and serological techniques [45,228]. Additionally, auto-antibodies, such as anti-cardiac myosin and other heart-specific antibodies, have been detected within patients with ongoing myocarditis and D C M , suggesting a link between autoimmunity and longterm disease [116,121,229]. Currently, there is no definitive evidence to prove that myocarditis leads to D C M but the utilization of animal models have suggested that there is a link [49,230,231]. 1.8 Treatment A t the current time, there is no definite treatment for myocarditis due to the unpredictability o f prognosis following myocarditis, and the treatment protocol should be 52 determined only after very careful analysis of the cause and clinical presentations. Supportive care is the basis of myocarditis management in patients since clinical presentations usually mimic flu-like symptoms and other cardiac disorders so the universally recommended therapy for myocarditis is bed rest and avoidance of physical exertion. Supplemental oxygen should be administered in all patients initially as pulmonary congestion may occur as a result o f cardiac dysfunction. Additional therapies aimed at limiting myocardial work and optimizing oxygen delivery to the tissues include fever reduction, management o f arrhythmias, and correcting anemia. For clinical symptoms of heart failure or arrhythmias, basic medications such as angiotensin-converting enzyme ( A C E ) inhibitors or angiotensin-receptor blocking agents, diuretics, beta-blockers, and anti-arrhythmic drugs (Amiodarone) should be administered. The use of ventricular assist devices, implantable cardiac defibrillators, intra-aortic balloon counterpulsation, and partial or complete cardiopulmonary bypass may be required i f cardiac dysfunction results in severely impaired cardiac ejection fraction and/or life threatening arrhythmias. Heart transplantation still represents the only definitive therapeutic option [4]. However, the onset and course of myocarditis is unclear and unpredictable, thus supportive management of myocarditis is not sufficient to prevent longterm cardiac damage. Therefore, specific therapies have been suggested to combat the varying clinical presentations and causes of myocarditis. Therapies currently under investigation include immunosuppression, immunoglobulin neutralization, and antiviral agents. 53 Figure 1.9: Outcomes of myocarditis (Adapted from Spotnitz and Lesch, 2006 [5]) In essence, this construct suggests four mechanistic pathways from acute myocarditis to sequelae. The level o f evidence supporting these pathways ranges from certain to unknown. Mechanism 1A Mechanism 1B Mechanism 2 Acute Massive Necrosis Acute DCM CHF/Death Timefraie Acute Acute Significant Necrosis Delayed DCM CHF (variable degree) Acute Carditis [+/- variable necrosis Delayed Intramyocardial dysfunction CHF (variable) (likely immune-mediated) Unknown Mechanism 3 t ^—> Acute Carditis Symptomatic or Asymptomatic Complete resolution^  with restiution of function and without res duai necrosis , New Onset DCM (see text) Chronic Status Proven Probably Hypothetical/ Hypothetical/ Proven Previously Postulated Proposed 1.8.1 Immunosuppression Immunosuppressive therapy has been employed in myocarditis patients, particularly in the later stages of the illness and therapeutic success was shown to be variable. Numerous uncontrolled, in which all o f the trial patients were given the treatment, and controlled, in which half o f the patients were given treatment and the other half were administered placebo, 54 immunosuppressive therapy trials have been reported during the past 2 decades for the treatment o f myocarditis. Therapies have included administration of corticosteroid prednisone alone, prednisone and azathioprine, prednisone and cyclosporine, as well as prednisone and O K T 3 (Ortho Biotech, Inc, Raritan, N J , U S A ) , which is a murine monoclonal antibody to the C D 3 receptor of human T-lymphocytes. The trials were performed in adults, children, as well as a combination of adults and children for treatment of inflammatory as well as dilated cardiomyopathy. Only a few randomized, placebo-controlled trials have been performed which examined the role of immunosuppressive therapy specifically in the treatment of acute myocarditis. In the first trial, Latham et al. [232] recruited 52 patients with early onset of D C M , of which only 12 patients presented with biopsy-confirmed myocarditis, and randomly assigned them to receive conventional therapy, which includes diuretics and other cardiac supportive medication, or conventional plus prednisone therapy for 3 months. The authors concluded that immunosuppression therapy did not improve survival as compared to conventional therapy and myocarditis was resolved in all patients regardless of treatment group. In a comparable study, 17 patients with active myocarditis, who were treated with a combination of prednisone and azathioprine for 3 months, were compared to 21 patients with comparable disease who underwent conventional treatment [233]. Despite an improvement in ejection fraction (EF) and NYHA-classif icat ion and a decrease in inflammation, both treatments augmented fibrosis and myocyte hypertrophy while the clinical outcome o f both groups was similar. Therefore, the authors also concluded that immunosuppression was determined to be ineffective in improving the primary end point in this study and had marginal clinical benefits in myocarditis patients. The Myocarditis Treatment Trial is thus far the largest randomized and controlled study o f 55 imrnunosuppression on myocarditis patients [234]. The study included 111 patients with histological evidence o f myocarditis and EFs under 45% from international centers and they were randomly assigned to three treatment groups: 1) azathioprine, prednisone, and conventional therapy; 2) cyclosporine, prednisone, and conventional therapy; and 3) conventional therapy alone for 24 weeks and follow-up was extended to 52 weeks. Unfortunately, prednisone and cyclosporine-based immunosuppressive therapy also produced no significant improvement in E F , survival, requirement for cardiac transplantation, and inflammatory activation. Despite the lack of clinical benefit o f immunosuppressive therapy in lymphocytic myocarditis, it has been postulated that patients presenting with giant cell myocarditis, which is often associated with an autoimmune process and frequently fatal, may benefit from high-dose immunosuppressive therapy and may improve survival in this population [12,235]. 1.8.2 High-Dose Intravenous Immunoglobulin Due to the ineffectiveness of immunosuppressive therapy, other trials have investigated alternative therapies. High-dose intravenous immunoglobulins (ivIG) therapy has been shown to be effective in managing myocarditis in animal models and moderate success in some clinical trials [236]. This type of therapy utilizes immunoglobulin (IG) preparations that have anti-microbial and anti-inflammatory effects, attributed to the interaction between the Fc portion of the IG and the low-affinity activating IgG Fc receptor, FcyRIII, leading to subsequent signal transduction through the receptor, though the exact mechanism is still unknown [237]. High-dose iv IG therapy has been shown to be effective treatment for a variety o f immunologically-mediated diseases such as Kawasaki's disease, 56 peripartum cardiomyopathy, giant cell myocarditis, and viral myocarditis [238-242]. In animal models of giant cell and viral myocarditis, IG treatment greatly enhanced survival and suppressed the development of disease as shown by reductions in immune infiltration and cardiac necrosis, possibly via downregulation of cytokines and inhibition o f immune cell activation, while increased ant i-CVB3 neutralizing antibodies and decreased viral titers in the myocardium were also observed in the viral models [239-242]. In clinical trials, the effectiveness of iv IG is still not entirely clear and results from ongoing studies, such as the double-blinded, randomized E S E T C I D (European Study on the Epidemiology and Treatment of Cardiac Inflammatory Disease) are still pending [35]. In non-randomized and non-controlled studies, the use of iv IG in addition to conventional therapy in 21 pediatric patients with suspected myocarditis demonstrated a trend for improved survival and cardiac function, as evidenced by smaller L V end-diastolic dimensions and improved fractional shortening in the treatment group as compared to historical controls [243]. Administration o f iv IG to six women with peripartum cardiomyopathy and three adult patients with histologically-confirmed myocarditis also showed that left ventricular E F was improved upon treatment [239,244]. In the only controlled and randomized study reported, McNamara et al. showed that immunoglobulin therapy does not provide benefit to patients with new-onset cardiomyopathy and myocarditis [245]. However, in this trial, o f the 62 recruited patients, only 10 had endomyocardial biopsy-proven myocarditis and the treatment resulted in no significant improvement in ejection fraction, functional capacity as assessed by metabolic stress testing, and number of post-treatment incidences. Despite the discouraging results from this study, successful treatment was shown in numerous case reports and non-controlled 57 studies; therefore, a randomized and placebo-controlled study with a larger cohort o f myocarditic patients is warranted. 1.8.3 A n t i - V i r a l One major reason why the treatment strategies described above produced inconsistent results is the fact that the causative agents of myocarditis was not taken into account. Different etiological agents may result in diverse disease progression, therefore, current investigations have now focused on targeting specific causative agents. The major form o f myocarditis is presented as lymphocytic myocarditis, frequently caused by viruses such as C V B 3 , so in cases o f biopsy-proven myocarditis patients with confirmed viral etiologies, treatment with anti-viral therapy may be beneficial and immunosuppression in these patients may be detrimental. Many studies have shown that the interferon anti-viral system can significantly decrease viral replication and dissemination in vitro and in vivo, and may be beneficial to patients with myocarditis specifically with detection of virus [90,84,103,240,246-248]. I F N treatment of mice infected with cytomegalovirus, encephalomyocarditis, or C V B 3 to induce myocarditis lead to reductions in necrosis, inflammation, and viral titers, possibly through downregulation of cytokines [248,250]. Thus, clinical trials have followed in this logic and investigated administration of I F N - a and IFN-p to myocarditic patients o f viral nature. Early non-randomized and non-controlled studies and case reports documented an improvement in cardiac function, as evidenced by a reduction in L V ejection fraction, pulmonary artery pressure, L V dimensions, and N Y H A functional class [251,252]. These encouraging results prompted larger, randomized, and placebo-controlled studies with longer follow-up timepoints. M i r i c et al. enrolled 180 patients to receive I F N - a , 58 thymomodulin (thymus-derived hormones with anti-inflammatory capabilities), or conventional therapy alone for 3 months and patients were examined up to 7 years after the end o f treatment [253]. L V ejection fraction, exercise tolerance, and mortality rate were significantly improved at all subsequent follow-up timepoint in patients treated with I F N - a and thymomodulin as compared to conventionally-treated patients. In subsequent studies by this same group, 40 and 38 patients presenting with myocarditis and/or D C M were randomized into three treatment groups: 1) conventional therapy plus I F N - a , 2) conventional therapy plus thymomodulin, and 3) conventional therapy alone. In both trials, the patients were examined up to two years following the end o f the treatment and endomyocardial biopsy, L V function by echocardiography, treadmill exercise test, Holter monitoring, and radionuclide assessment of left ventricular function during exercise were evaluated [254,255]. L V ejection fraction, L V reserve, and maximum exercise time were significantly increased during follow-ups in a higher percentage of the I F N - a and thymomodulin-treated patients as compared to controls. The E K G was normalized in a majority of patients who received I F N - a or thymomodulin treatment and they also had significant improvement in their N Y H A functional class [255]. However, these trials did not account for viral presence and the number of patients with myocarditis as compared to D C M was low. In the highly anticipated E S E T C I D , a double-blind, randomized, placebo-controlled trial, 182 patients with ongoing inflammatory processes and L V ejection fraction <45% were randomized into 4 treatment groups: 1) a combination of prednisolone and azathioprine; 2) IFN-a; 3) iv IG; and 4) conventional therapy [35]. This study compares all the current therapies for myocarditis and its final results w i l l provide more comprehensive information on the effects o f each treatment. 59 The emerging concept on management of myocarditis is tailoring therapy to the specific causes of disease because the disease progression is highly variable. For example, patients presenting with viral myocarditis w i l l not benefit from immunosuppressive drugs, since this diminishes the ability of the immune system to eliminate the offending virus. Further, it is recommended to administer immunosuppressive drugs and iv IG to patients who likely suffer from an autoimmune-mediated disease, such as giant cell myocarditis, and anti-viral therapy, such as I F N treatment, is more beneficial to patients with viral myocarditis (Table 1.7). Thus, recognizing myocarditis at its early stages by honing the diagnostic tools to differentiate the different forms of myocarditis and tailoring the treatment to each form give the clinicians the power to best manage this potentially debilitating disease. 60 Table 1.7: Myocarditis diagnosis and treatment strategies (Adapted from Maisch et al. [236]). The gold standard for diagnosis o f myocarditis is via analysis o f endomyocardial biopsy samples by pathologists, as guided by the recommendations of the new W H O / W H F classification (Table 1.3). Treatment is then administered based on the etiological agent. Diagnostic Approach: Symptomatology: precordial discomfort, dyspnoea, rhythm disturbance (VT, Vfib, VES) +/- echocardiography: disturbed global or segmental contraction and relaxation, pericarditis (Horowitz classification B to D) + coronary angiography: exclusion of CAD or functional disturbance not explained by CAD Inflammation (biopsy) >14 ly & Mo/mm* PCR for cardiotropic agents positive ~ virus positive myocarditis 1 Agent specific antiviral therapy Endomyocardial biopsy Inflammation (biopsy) >14 ly & Mo/mm2 PCR for cardiotropic agents negative = autoreactive myocarditis T Immunosuppressive or immunomodulatory therapy Inflammation (biopsy) <14ly& Mo/mm* PCR for cardiotropic agents positive - viral heart disease Agent specific antiviral therapy Inflammation (biopsy) <14ly&Mo/mmJ PCR for cardiotropic agents negative = no myocarditis Heart failure and antiarrhythmic therapy 61 1.9 Rationale. Hypothesis, and Experimental Aims 1.9.1 Rationale Myocarditis is a highly variable disease and may lead to dilated cardiomyopathy or heart failure. However, the mechanisms governing the pathogenesis of myocarditis and the progression to end-stage disease are controversial and require further investigation. Following viral injury to the heart, many complex reparation processes are stimulated and imbalance of any one of the steps w i l l lead to chronic disease. Virus infection stimulates the immune response to eliminate the virus and to regulate the reparation process, which includes accumulation of matrix and cellular proliferation within myocyte dropout regions. The fibrotic process and the subsequent remodeling is crucial in maintaining proper cardiac structure and function, due to the low frequency of cardiomyocyte division and the importance of contractile function maintenance. Matrix metalloproteinases ( M M P s ) are major degraders o f the matrix and regulators o f the wound healing process, since they modulate migration of immune cells and fibroblasts, angiogenesis, as well as activation o f cytokines and growth factors. They have been shown to play major roles in other cardiac diseases, such as M I and hypertrophy, and inhibition o f certain M M P s during specific timepoints following cardiac injury significantly alters the course of the disease. However, the role of M M P s during viral myocarditis has not been elucidated. A s such, in this dissertation, I investigate the expression profile of select M M P s and their inhibitors, TIMPs , following C V B 3 infection at various timepoints using a well-established mouse model. I also use genetically modified mice in order to determine the effects o f specific M M P deficiency on pathogenesis of myocarditis. These experiments provide valuable insight into understanding the role o f M M P s in myocarditis. 62 1.9.2 Hypothesis Dysregulation of M M P s and TIMPs during C V B 3 infection promotes viral replication and the progression of chronic myocarditis through modulation of inflammation and E C M remodeling. 1.9.3 Specific Aims 1. To characterize M M P - 2 , M M P - 8 , M M P - 9 , M M P - 1 2 , M M P - 1 3 , M T 1 - M M P , and their endogenous inhibitors, the TIMPs, in coxsackievirus-infected mouse hearts at days 3, 9, and 30 post-infection. 2. To determine the mechanisms by which M M P s act to affect the infected heart. 3. To examine the relationship between inflammation and E C M alteration in C V B 3 -infected hearts. 1.9.4 Methodology Overview I used P C R , immunohistochemistry, and gelatin zymography to determine the transcriptional, translational, and activation status of select M M P s following C V B 3 infection of mice. To determine the effects of the specific M M P s in this disease, I infected genetically deficient mice lacking M M P - 1 2 , M M P - 8 , and M M P - 9 and performed histopathological assessments and determined the inflammatory response by evaluating the amount of immune cells, using immunohistochemistry, and cytokines, using quantitative real-time P C R , in the myocardium o f these mice and compared these results to W T counterpart mice. Finally to examine the relationship between inflammation and matrix alterations, I inhibited the 4-IBB pathway, which provides co-stimulation in T-cell activation, and determined the functional, 63 using echocardiography, and matrical changes in the heart. The experimental scheme is shown in Figure 1.10. Figure 1.10: Exper imental design CVB3 A/J mice (10s PFU, ip) Sham or Virus Infection 1 3d, 9d and 3()d pi 1 PCR for MMP-2, MMP-9, & MMP-12 mRNA Immunohistochemistry for MMP-2, MMP-9, & MMP-12 Western blot for MMP-8, MMP-13, MT1-MMP, & TIMPs Gelatin Zymography for assessment of MMP-2 & MMP-9 activity levels HL-1 and MMP-12KO mice *'J> (10s PFU, ip) (10s PFU, ip) I*? (10s PFU, ip) MMP-8KO and MMP-9KO mice A/J mice sham+ vehicle, sham+ anti-4-lBBL virus+ vehicle, virus+ anti-4-1 BBL • Viral plaque assay • Immunoblot for MMP-12 * Histological examination • Matrix assessment via picrosirius red & Mo vat's pentachrome stains * Viral detection * Apoptosis assessment • Histological examination a • Matrix assessment via a picrosirius red staining • Immunohistochemistry for , CD45, PMN & CD3 • quantitative PCR for cytokines Heart function - echocardiography Histological examination Matrix assessment via picrosirius red staining PCR for MMP-2, MMP-9 & MMP-12 Immunohistochemistry for CD45 & CD3 (inflammatory cells) 64 1.9.5 Potential Significance of Findings M y results have provided insight into the roles of M M P s in a virus infection model. Previously, extensive studies have focused on the roles of M M P s in compensatory, adaptive cardiac diseases, such as myocardial infarction and hypertrophy, whereas my investigations explored the activities o f M M P s in microbial-induced diseases and the ensuing inflammatory response. In contrast to previous reports, in which M M P s were shown to contribute to the progression of disease and that inhibition of these enzymes ameliorated or diminished the symptoms, I found that M M P s have a protective role in virus infection and that deficiency in select M M P s , such as M M P - 9 and M M P - 1 2 , greatly increased the severity o f the disease, possibly through improper immune response activation. 65 1.10 References 1. Sobernheim JF (1837). Praktische Diagnostik der inneren Krankheiten mit vorzueglicher Ruecksicht auf pathologische Anatomie. Hirschwald, Berlin, p 117. 2. Fiedler K L A . Uber akute interstitielle Myokarditis Zentralblatt fur innere Mediz in 1900;2:212-213. 3. 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Heart 1996;75:596-601. 86 CHAPTER II - EXPRESSION PROFILE OF MMPs AND TIMPs IN COXSACKIEVIRUS-INDUCED MYOCARDITIS 2.1 Rationale In the mouse model of viral myocarditis, both direct virus-mediated cytolysis and immune cell-mediated injury result in extensive damage to the heart and triggers profound extracellular matrix ( E C M ) remodeling which may ultimately lead to D C M . M M P s are major regulators of inflammation, cardiac repair, and E C M remodeling. Previous studies have shown upregulation of M M P - 8 and M M P - 9 with concomitant downregulation of T I M P -1 following C V B 3 infection but more comprehensive profiling of this class of protease and their inhibitors have not been performed before. There are over 23 M M P s with four associated endogenous inhibitors: T IMP-1 , TIMP-2 , T IMP-3 , and TIMP-4 [1]. I chose to examine M M P - 2 , M M P - 8 , M M P - 9 , M M P - 1 2 , M M P - 1 3 , and M T 1 - M M P in this study because these proteases have been shown extensively to participate in pathogenesis following M I , pressure and overload-induced hypertrophy, and vascular diseases [2-4]. Previous unpublished studies using microarray-based methodologies in our laboratory have' found a significant increase in the expression of M M P - 1 2 , or metalloelastase, in the setting o f C V B 3 -induced myocarditis (Figure 2.1). Here I further characterized the activation and expression o f M M P s and TIMPs in viral myocarditis. These enzymes have a large variety o f substrates, including collagen types I, III, and IV as well as elastin and fibronectin, all o f which have been shown to be important during pathogenesis and repair of the myocardium following C V B 3 infection [1]. It has been postulated that these enzymes not only degrade the matrix A version of this chapter has been published. Cheung C et al. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in coxsackievirus-induced myocarditis. Cardiovasc Pathol, 2006; 15(2): 63-74. 87 but also regulate inflammation by processing cytokines, such as IL-l (3 and TGF-P . The spatial and temporal balances of MMPs and TIMPs are very important in pathogenesis and repair, since early activation of MMPs may be beneficial in viral clearance while chronic activation may provoke maladaptive remodeling. Therefore, to determine the contribution of these MMPs in viral myocarditis, we evaluated their transcriptional and translational expression as well as localization within the myocardium following coxsackievirus infection. Figure 2.1: Affymetrix microarray analysis of MMPs. MMP, TIMP, and A D A M transcriptional expression were analyzed using the Affymetrix oligonucleotide microarray platform in CVB3-infected mouse hearts at 3, 9, and 30 days post-infection. MMP-12 was highly upregulated. Days Post-Infection 30 % TIMP-1 - A D A M - 8 MMP-3 MMP-12 o 0 88 2.2 Materials and Methods 2.2.1 Virus Preparation and Plaque Assay y i ru s stocks were propagated by passage in HeLa cells and viral titers were determined by a standard plaque assay procedure [5,6]. A t least 1X10 6 plaque forming units (PFU) of virus were applied to a confluent monolayer of HeLa cells grown in a T75 flask. After 24 hours, all o f the HeLa cells were lysed by the virus and the medium was collected and centrifuged at 2000 rpm for 10 minutes to remove cell debris. The supernatant containing the virus was aliquoted and stored at -80°C. In order to determine the viral titer, the supernatant was serially diluted by orders of ten in 200iiL of serum-free Dulbecco's Modified Eagle medium ( D M E M from Invitrogen, Burlington, ON) and applied to H e L a cells grown in 6-well plates for 1 hour at 37°C. After removal of the virus, the cells were washed twice with phosphate buffered saline (PBS) and two milliliters (mL) o f fresh D M E M , containing 10% fetal bovine serum (FBS, Invitrogen) and 0.75% agar (Sigma-Aldrich, Oakville, ON) , was overlaid over the infected cells followed by incubation at 37°C for 48-72 hours. The cells were then fixed with Carnoy's fixative (25% acetic acid, 75% ethanol) and then stained with 1% crystal violet (Sigma-Aldrich). The assay was performed in triplicate to ensure consistency in the serial dilutions. This method essentially allows virus to propagate but not disseminate since the virus could not spread through the agar; thus virus-infected cells were lysed and created clear plaques devoid of cells after staining with crystal violet, which stains lipids on viable cells. Vi ra l titer was calculated and determined as P F U per milliliter. 89 2.2.2 Virus Infection of Animals Five week-old male A / J mice (Jackson Laboratories, Bar Harbor, M E ) were injected intraperitoneally (i.p.) with 200uL of C V B 3 (Gauntt strain, 10 5 P F U ) or P B S , and then sacrificed at 3, 9, and 30 days pi . Five to twelve mice were sacrificed and harvested at each timepoint. A l l animal procedures were in accordance with the Animal Care Committee, University of British Columbia. 2.2.3 Quantitation of Viral Titer Heart tissue was homogenized in serum-free D M E M using glass mortar and pestle (Kontes Glass, Vineland, NJ) and then assessed using plaque assay for quantitation of virus titer. In situ hybridization (ISH) was performed on heart sections to probe for positive and negative viral genomic strands, as previously described [7]. Briefly, paraffin-fixed tissue was hybridized with digoxigenin-labeled C V B 3 antisense R N A probes, which were prepared from the full-length C V B 3 c D N A using an in vitro transcription kit according to the manufacturer's instructions (Promega, Madison, W l ) . Hybridized riboprobes were detected using the ABComplex kit with an alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Diagnostics, Mannheim, Germany) and Vector Red colour substrate (Vector Laboratories, Burlingame, C A ) . The stained sections were counterstained with hematoxylin and images were captured using a Nikon inverted microscope and Spot digital camera. 2.2.4 Histological Assessment Mid-ventricular myocardium was harvested and submitted for fixation in 10% neutral-buffered formalin (Sigma-Aldrich) followed by paraffin embedment. Histological sections were stained with hematoxylin and eosin ( H & E ) , to detect areas o f myocardial 90 damage and inflammatory infiltration, and with picrosirius red for collagen quantitation and architecture. Tissue sections were dewaxed, rehydrated, and processed as follows: 1. hematoxylin stain, 5 minutes 2. rinse with dr/bO, 1 minute 3. 1 % acid alcohol, 5-10 seconds 4. rinse with dl-fiO, 1 minute 5. 1% lithium chloride, 30 seconds 6. rinse with dH^O, 1 minute 7. 70% isopropanol, 30 seconds 8. 1% eosin in 80% ethanol, 30 seconds 9. air-dried and coverslipped. For the latter stain, paraffin-embedded sections were rehydrated, stained for 1 hour i n 0.1% Sirius-red solution (Direct Red 80, Sigma-Aldrich) in saturated picric acid, rinsed in running tap water without counterstaining, air-dried, and coverslipped. H&E-stained sections were graded blindedly by a cardiovascular pathologist ( B . M . M . ) on the following scale: 0, no or questionable presence; 1-2, limited focal distribution; 3-4, intermediate severity; and 5, coalescent and extensive foci over the entirety of the transversely sectioned ventricular tissue. Images were all captured using a Nikon inverted microscope and Spot digital camera. 2.2.5 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) R T - P C R was used to detect the transcript levels of M M P - 2 , -9, and -12 after virus infection. Total mouse R N A was isolated from the basal portion of the heart using RNeasy kit (Qiagen, Valencia, C A ) and 0.5|ig of R N A was converted to complementary 91 deoxyribonucleic acid ( c D N A ) using Superscript reverse transcriptase (Invitrogen) according to manufacturer's protocol. The P C R primers and conditions are summarized in Table 2.1. A s a control, 18S ribosomal R N A (rRNA) primers were purchased from Ambion (Austin, T X ) and performed according to manufacturer's protocol. We performed assays on 4 mice from each timepoint. Table 2.1: PCR primer sequences and conditions for detection of MMPs. Transcriptional expression of M M P - 2 , M M P - 9 , and M M P - 1 2 were analyzed by P C R in CVB3-infected mouse hearts . Gene Primers PCR Conditions Size 0>P) Genbank MMP-2 (forward) 5' G G C C A T G C C A T G G G G C T G G A 3' (reverse) 5' C C A G T C T G A T T T G A T G C T T C 3' 35 cycles 94°C Imin 57°C Imin 72°C Imin 762 NM_008610 MMP-9 (forward) 5' C T A C T C T G A A G A C T T G C C G 3' (reverse) 5' C C A T A C A G T T T A T C C T G G T C 3' 35 cycles 94°C Imin 57°C Imin 72°C Imin 881 NM_013599 MMP-12 (forward) 5' A G A G G T C A A G A T G G A T G A A G 3' (reverse) 5' T A T A T G C T C C T G G G A T A G T G 3' 40 cycles 94°C Imin 51°C Imin 72°C Imin 792 NM_008605 2.2.6 Immunohistochemistry Paraffin-embedded heart sections were sectioned 3-4|im in thickness and probed for the presence and localization of M M P - 2 , M M P - 9 , and M M P - 1 2 using immunohistochemistry. The slides were dewaxed, rehydrated, microwave-heated in 6% urea for 10 minutes, and incubated overnight at room temperature using 1/500 ant i -MMP-2 (Sigma-Aldrich), 1/1000 ant i -MMP-9 (Sigma-Aldrich), and 1/50 ant i-MMP-12 (Chemicon) polyclonal antibodies diluted in serum-free protein blocking reagent (Dako). The sections were then washed with tris base saline (TBS) pH7.6 and 1/200 anti-rabbit secondary antibody (Dako) was applied for 1 hour at room temperature. After washing, staining was amplified and exposed using the ABComplex system (Dako, Mississauga, ON) in conjunction with the substrates 3,3'-diaminobenzidine [ D A B ] (Dako) for M M P - 9 detection and Vector Red (Vector Labs) for M M P - 2 and M M P - 1 2 detection. Images were all captured using a Nikon inverted microscope and Spot digital camera. Five to eight animals from each timepoint were used in this assay. 2.2.7 Immunofluorescent Co-Localization Mid-ventricular sections were dewaxed, rehydrated, treated with 0 .2M glycine for 10 minutes at room temperature, and double-stained with 1/25 CD45 antibody (pan hematopoietic cell marker, B D Biosciences, San Jose, C A ) in conjunction with M M P - 2 , M M P - 9 , or M M P - 1 2 antibodies, using the conditions as described above. The secondary antibody to the CD45 antibody was goat anti-rat IgG2a conjugated to Alexa Fluor® 488 (Molecular Probes/Invitrogen, Burlington, ON) and goat anti-rabbit IgG F(ab')2 conjugated to Alexa Fluor 594 was used for detection of all M M P s . Hoechst dye (Molecular Probes/Invitrogen) was used to stain the nuclei and images were captured using confocal microscopy with the Leica SP2 A O B S microscope. Five to eight animals from each timepoint were used in this assay. 2.2.8 Gelatin Zymography Heart tissue was assessed using gelatin zymography to detect the activity of M M P - 2 and -9, as previously described [8]. Frozen apices were homogenized in 0.1% Nonidet P-40 (NP-40) buffer with glass homogenizers (Kontes), the protein concentrations were measured using the B C A protein assay kit (Pierce Chemical Company, Rockford, IL) according to manufacturer's protocol, and then 100|J,g of total protein was electrophoresed under non-93 reducing conditions through 7.5% Laemmli acrylamide gels, containing 0.05% porcine gelatin (Sigma-Aldrich) as substrate, at 100V for 2 hours. Non-reducing conditions is defined as electrophoresis of non-denatured proteins by omission o f P-mercaptoethanol and heat inactivation of the proteins. Gels were washed twice with 5% Triton-X-100, incubated in activation buffer (CaCl2, Tris pH8.0, and sodium azide) for 48 hours at 37°C, and stained with Coomassie brilliant blue (Biorad, Hercules, C A ) . Purified enzymes purchased from Chemicon (Temecula, C A ) and homogenized spleen tissue, which have high baseline M M P -9 and M M P - 2 activities, were activated with I m M aminophenylmercuric acetate ( A P M A ) overnight at 37°C and run alongside samples as positive controls [9]. Five to eight animals from each timepoint were used in this assay. 2.2.9 W e s t e r n B l o t M M P - 8 , M M P - 1 3 , M T 1 - M M P , and TIMPs were detected using immunoblotting assay. Frozen apices were homogenized in 0.1% NP-40 in P B S buffer with a glass homogenizer and lOOug of total protein was electrophoresed under reducing conditions (which included addition o f P-mercaptoethanol to the protein samples and boiling the mixture for 10 minutes), through 7.5% ( M M P s ) or 10% (TIMPs) polyacrylamide gels at 100V for 2 hours. The proteins were transferred overnight onto nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ) using a M i n i Trans-Blot cell (Biorad) at 30V. The membranes were then blocked with 5% skim milk powder in T B S with 0.1% Tween 20 (TBS-T) p H 7.6, and the following antibodies and dilutions were applied overnight at 4°C in 2.5% skim milk buffer: 1/1000 polyclonal M M P - 8 (Chemicon), 1/1000 polyclonal M M P - 1 3 (a generous gift from Dr. Chantal Peeters-Joris, Laboratoire de Chimie Physiologique, Universite catholique de Louvain, Louvain, Belgium), 1/1000 polyclonal 94 a n t i - M T l - M M P (Chemicon), 1/4000 polyclonal TIMP-1 (Affinity BioReagents, Golden, CO) , 1/200 monoclonal TIMP-2 (Oncogene, San Diego, C A ) , 1/2000 polyclonal TIMP-3 (Sigma-Aldrich), and 1/1000 polyclonal TIMP-4 (Neomarkers/Lab Vis ion , Fremont, C A ) antibodies. Detection of the Western blots was performed using the H y p e r E C L system (Amersham) according to manufacturer's protocol. Glyceraldehyde-3-phosphate dehydrogenase ( G A P D H ) antibody was purchased from Research Diagnostics (Concord, M A ) and was used as a control. Four to twelve animals were used in these assays. 2.2.10 Data and Statistical Analyses The National Institutes of Health (NIH) ImageJ 1.3 l v program (http://rsb.info.nih.gov/ij/) was utilized to quantitate Western blot and zymography band intensities. Corrected values were then compared to sham and expressed as a ratio o f infected samples to sham or fold change over sham. The data are presented as mean ± standard deviation (SD) in the text and graphs. Statistical analyses were performed using the SPSS 14.0 (SPSS Inc., Chicago, IL) software program. Pairwise comparisons between sham and virus-infected samples were conducted with a multiple comparison method, the two-sided Dunnett's test, using sham as reference level and differences were considered significant at p<0.05. 2.3 Results 2.3.1 Virus Detection and Histological Examination Plaque assay and in situ hybridization were performed to determine the presence and amount of virus within CVB3-infected hearts. A s early as 3 days p i , considerable amounts o f viral particles were detected, using plaque assay, within the myocardium which remained 95 high up to day 9 pi and even at day 30, low levels of virus persisted (Figure 2.2). Consistent with previous experience, both positive and negative (not shown) viral genomic sequences were detected in CVB3-infected hearts up to 9 days pi but were not detected thereafter (Figures 2.3B-D), due to less sensitivity of ISH as compared to plaque assay. A s early as 3 days pi , small foci of infected myocytes were observed (Figure 2.3B, arrows), and at this timepoint viremia and cytopathic effects (Figure 2.3F, arrowhead), including vacuolization and presence o f contraction bands, were detected without immune infiltration. A t day 9, widespread infection of cardiocytes (Figure 2.3C) and immune infiltration (Figure 2.3G) were detected throughout the heart muscle. Leukocytes localized to areas of viral genome positivity and were probably involved in virus clearance, resulting in myocytolysis, calcification of heart tissue, and other structural damages to heart muscle. B y 30 days p i , both viral propagation and inflammation had subsided and the areas o f myocyte dropout had begun to be transformed into scar tissue, thus signalling the resolution o f the myocarditic phase of disease (Figures 2.3D and 2.3H). Picrosirius red stains collagens with high affinity and was used to detect fibrosis. The fibrillar collagen types I and III are most abundant in the adult heart. Virus-infected hearts stained with picrosirius red revealed a marked increase in collagen deposition beginning from 9 days p i , as well as soft tissue distortion (Figure 2.3K). Sham- and CVB3-infected heart tissues at day 3 pi (Figures 2.3I-J) were similar and showed no differences in perivascular or interstitial collagen amounts and organization. A t 9 days pi , collagen deposition accumulated adjacent to damaged, dying cardiomyocytes with a slight increase in perivascular collagen (arrows). Extensive myocyte dropout and calcification were evident, and replacement fibrosis appeared, providing the heart with substitute attachment sites for surviving cells 96 possibly salvaging contractile function. However, by 30 days pi, much of the dead tissue had been removed and scar formation was in progression (Figure 2.3L). Replacement fibrosis progressed to prominent collagen deposition, focal scars, and perivascular matrix accumulation. Figure 2.2: Plaque assay quantitation of viral particles. Quantitation of viral titer was performed by a standard plaque assay technique following CVB3 infection at 3, 9, and 30 days pi. N=5-12 mice per timepoint. The viral load in the myocardium peaked at 9 days pi but was still detected at low amounts at day 30. 3 9 30 Days Post-Infection 2.3.2 Transcriptional Regulation of MMPs I then examined the MMPs, which are known to be involved in the normal turnover of the E C M as well as major remodeling events in the heart. I first characterized the transcriptional levels of MMP-2, -9, and -12 in the mouse hearts after virus infection using RT-PCR. Figure 2.4 shows that the expression of all three MMPs was upregulated in the myocardium following infection as compared to control. MMP-2, MMP-9, and MMP-12 transcripts 97 peaked at 9 days pi and the differences from baseiine were all statistically significant (p=0.015 for M M P - 2 ; /XO.0001 for M M P - 9 ; and pO.OOOl for M M P - 1 2 transcripts). Expression o f M M P - 1 2 remained significantly increased at day 30 pi (pO.OOOl) and upward trends were seen for M M P - 2 and M M P - 9 . Figure 2.4B shows the graphical representation o f the densitometric analyses. 98 Figure 2.3: Analysis of the viral genome and histological assessment of the myocardium by H & E and picrosirius red staining. (A-D) In situ hybridization staining for positive viral genomic R N A of sham- and CVB3-infected mouse hearts at 3, 9, and 30 days pi . (E -H ) H & E staining of sham- and CVB3-infected heart tissue at days 3, 9, and 30. (I-L ) Fibrillar collagen amount and orientation were detected by picrosirius red staining of the heart under polarized light microscopy. Bar in image=50^un. ISH H&E Picrosirius Red 99 Figure 2.4: Transcriptional analysis of MMP-2, MMP-9, and MMP-12. (A) R T - P C R for M M P - 2 , -9, and -12 in sham- and CVB3-infected mouse hearts at 9 and 30 days pi . (B) Densitometric analyses of the bands (mean±SD). A n individual sample was run per lane and the intensities from each lane were calculated as a ratio of infected to sham or fold change over sham, where sham is equal to one ( M M P - 2 and M M P - 1 2 ) . M M P - 9 is expressed as intensity in arbitrary units. N=5-12 for each enzyme. * p O . 0 0 0 1 , % /K0.02 Days Post-Infection Days Post-Infection MMP-12 PCR S H A M 9 30 Days Post-Infection 100 2.3.3 Protein Expression and Localization of MMPs Protein expression and localization were then determined by immunohistochemistry. Figure 2.5A shows M M P - 2 positivity within cardiomyocytes and vasculature in sham hearts. Immunopositivity was diffuse and found in both ventricles as well as in the ventricular septum. A t 9 days pi (Figure 2.5B & 2.6D), there was a dramatic increase in M M P - 2 staining within myocarditic hearts, particularly in and around necrotic foci, but also in resident cardiac cells of normal regions. Immunofluorescent co-localization staining for CD45 (Figure 2.6B) and M M P - 2 showed that the majority of M M P - 2 immunopositivity (Figure 2.6C) at day 9 was in the extracellular matrix and in cardiomyocytes (arrowheads) with rare immune cell co-expression (Figure 2.6D, orange staining, arrows). A t 30 days p i , evidence of substantial scar tissue deposition reflects myocardial reparation. M M P - 2 immunopositivity within these hearts had generally decreased to basal levels, but strong staining was localized to the rare scar foci (Figure 2.5C). Figures 2.5D and 2.5E illustrate minimal M M P - 9 positivity in normal and early-infected myocardium. A t day 9 (Figures 2.5E & 2.6G), a slight increase in M M P - 9 expression, localizing to immune cells (Figures 2.6G-H, arrows) and necrotic regions was seen, but rarely in cardiac resident cells. In the healing myocardium (Figure 2.5F), M M P - 9 expression had returned to baseline levels, and remaining immunopositivity was either within rare infiltrating leukocytes or apparent transformed fibroblasts. I detected constitutive expression of metalloelastase within sham hearts, although the signal was relatively weak (Figure 2.5G). This is the first time, to my knowledge, that M M P -12 has been detected within normal hearts and localized to resident cardiac cells. B y 9 days pi (Figures 2.5H & 2.6K), there was a significant increase in staining within the myocarditic 101 heart and this staining was strong within the necrotic foci as well as in remote regions. Few CD45-positive cells expressed colocalization to M M P - 1 2 (Figure 2.6J & 2.6L) while the majority o f metalloelastase immunoreactivity appears to be localized to cardiomyocytes or E C M , which remains to be elucidated. B y day 30, infected hearts still expressed increased staining for M M P - 1 2 as compared to sham, but the staining was much weaker than day 9 hearts. Metalloelastase was detected in a patchy distribution in day 30 hearts and was found within bundles of myofibers throughout the ventricle walls and septum. Figure 2.5: Protein expression and localization of MMPs-2, -9, and 12. Immunohistochemistry for (A-C) M M P - 2 , ( D - F ) M M P - 9 , and (G-I ) M M P - 1 2 in C V B 3 - and sham-infected mouse hearts at 9 and 30 days pi . (A) Sham and day 3 [not shown] infected hearts showed diffuse positive staining for M M P - 2 especially within the vasculature. (B) A t 9 days p i , there was a dramatic increase in M M P - 2 immunopositivity within resident cardiac cells as well as within necrotic foci. (C) B y day 30, the immunopositivity for this protease had diminished to basal levels but M M P - 2 could still be detected within scars. (D) M M P - 9 was not detected in sham mouse hearts. (E) However, at day 9, there was an increase in M M P - 9 immunopositivity, correlating with immune infiltration and often localizing to inflammatory cells. (F) B y day 30, immunopositivity subsequently decreased and was rarely detected. (G) M M P - 1 2 was detected in sham hearts in a diffuse pattern. (H) Immunopositivity for this protease increased at day 9 pi and could be detected within resident cardiac cells as well as necrotic foci. (I) B y day 30, the staining decreased below 9 day levels but was still increased as compared to sham. Negative rabbit IgG controls are shown for both (J) vector red and (K) D A B staining. Bars in image=50|im. A t least four animals were analyzed at each timepoint. 102 MMP-2 MMP-9 MMP-12 103 Figure 2.6: Immunofluorescent co-staining for CD45 and MMPs in virus-infected hearts at 9 days pi. (A-D) Double-staining for CD45 and M M P - 2 was performed and viewed using confocal microscopy. In accordance to Figure 2.5, there was intense M M P - 2 immunopositivity (arrowhead) within cardiomyocytes and in the E C M . There was rare co-localization of M M P - 2 and CD45 (orange, arrows). (E-H) M M P - 9 co-localized mostly to immune cells (orange, arrows) and was rarely found in the myocardium. (I-L) M M P - 1 2 was abundant mostly in the myocardium and was rarely associated with CD45-expressing cells. (A,E,I) Blue staining denotes D A P I nuclei staining, (B,F,J) green detects CD45, and ( C , G , K ) each M M P is shown as red staining. (D,H,L) A l l three channels are overlaid in these figures and co-localized proteins are represented as orange. Images were taken with a 63X objective lens. N=4-8 animals for each M M P . M M P - 2 M M P - 9 M M P - 1 2 A E I B F J C G K D H L 104 Immunoblotting was used to detect the presence and amount of M M P - 8 , M M P - 1 3 , and M T 1 - M M P , which are the major collagenases in mice. Different sources o f M M P - 8 produces different forms and sizes of this enzyme, notably PMN-derived p roMMP-8 exists as an 85 k D a proprotein that is cleaved to a 75 k D a active form while mesenchymal-derived p r o M M P - 8 is first produced as a 55 kDa molecule that is further degraded to several lower molecular weight active forms. A s shown in Figure 2.7A, I detected at least 3 major forms o f M M P - 8 , the PMN-derived proform, mesenchymal-derived p roMMP-8 , and an active mesenchymal enzyme. A l l three forms were not altered at 9 days pi in CVB3-infected mice but by 30 days pi , the M M P - 8 enzymes were substantially decreased as compared to sham, notably the active mesenchymal-derived enzyme, although this did not reach statistical significance (p=0.1). Only the proform o f M M P - 1 3 (60 kDa) was detected and there was no significant change in the protein expression of M M P - 1 3 at any of the investigated timepoints (Figure 2.7). In contrast, M T 1 - M M P was detected as a 64 kDa proform and 50 k D a active enzyme. In a biological system, M T 1 - M M P is first produced as a zymogen and then this protease undergoes intracellular activation via furin cleavage within the trans-Golgi network [1]. The active M T 1 - M M P is then trafficked to the cell membrane. I observed an increase in cell-membrane bound active M T 1 - M M P at 9 and 30 days p i , with a corresponding decrease in the intracellular proform (Figure 2.7A), although this did not reach statistical significance (p=0.l). Pictoral representation o f the densitometric analyses of the protein expressions o f these M M P s are displayed in Figure 2.7B. 105 Figure 2.7: Immunoblotting analysis of MMP-8, MMP-13, and MT1-MMP. (A) Representative images showing immunoblot detection for M M P - 8 , M M P - 1 3 , and M T 1 -M M P . G A P D H was used as endogenous control and N=4 mice were used to each enzyme at each timepoint. (B) Densitometric analyses was performed and graphically represented. A l l of the blots were normalized to G A P D H and the graphs are shown as a ratio of infected to sham (fold change over sham) where sham is equal to one (mean±SD, N=3-5) A 85, 60' 50' 40 ' 25 ' Days Post-Infection SH 9 30 SH 9 30 SH 9 30 SH 9 30 , V . . v M M P - 8 MMP-13 M T 1 - M M P G A P D H B S H A M 9 30 Days Post-Infection | proMMP-8 (PMN) ~2 proMMP-8 (mesenchyme) | j active M M P - 8 (mesenchyme) I S H A M 9 30 Days Post-Infection MMP-13 S H A M 9 30 Days Post-Infection | p r o M T l - M M P ~] active M T 1 - M M P 106 2.3.4 Gelatinolytic Activity of MMP-2 and MMP-9 I then investigated the gelatinolytic activity of M M P - 2 and M M P - 9 using zymography. The proform and active enzyme of M M P - 2 were detected in all samples, whereas only the lOOkDa band of M M P - 9 was visualized in this assay. A s shown in Figure 2.8, the gelatinolytic activities of both proteases began to increase as early as 3 days pi , but only upregulation of M M P - 9 (p=0.02) reached statistical significance. During the inflammatory phase, the levels of p roMMP-2 (p=0.004), total M M P - 2 (p=0.002), and M M P -9 activity (pO.OOOl) were significantly upregulated as compared to sham. However, both M M P s subsequently returned to basal levels during the late stage of infection at day 30. 2.3.5 Protein Expression of TIMPs To determine the possible mechanisms of increased M M P activities, I examined the protein expression of the endogenous inhibitors of M M P s , TIMPs, by immunoblot assay. The four T IMPs can inhibit all species of M M P s with K i values ranging from subfemtomolar to nanomolar [10]. TIMP-2 is unique in that it aids in activation (in conjunction with M T 1 -M M P ) o f M M P - 2 and inhibits this enzyme with one of the lowest dissociation constants [10]. Figure 2.9 shows constitutive protein synthesis of all four T IMPs in sham hearts. TIMP-1 and TIMP-2 expression was essentially unchanged throughout the infection. TIMP-3 and TIMP-4 were produced in large amounts in sham hearts and I show here that both TIMPs were decreased following virus infection. TIMP-3 was substantially and significantly downregulated at days 3 (p=0.002) and 9 (pO.OOOl), during the acute phase of the infection. T I M P - 4 was decreased on day 9 (p=0.04). Taken together, these results suggest that regulation of M M P s in myocarditis may occur both at the transcriptional and post-translational level, v ia T I M P inhibition. Since 107 TIMP-1 and TIMP-2 were relatively unchanged during the infection, these inhibitors may not play as large a role as the other two TIMPs. Figure 2.8: Zymography analysis of MMP-2 and MMP-9. (A) Representative image of gelatin zymography and (B) densitometric analyses of each band. Both MMP-2 and MMP-9 increased and peaked at 9 days pi. N=5-12 animals per timepoint. * /?<0.0001, # /?<0.005, % /?<0.02. Molecular ^ Weights Sham Days Post-Infection lOOkDa 70kDa 67kDa 60kDa B e a ea .a U -a "5 6 M M P - 9 M M P - 2 • Pro M M P - 2 • Active M M P - 2 • Total M M P - 2 Days Post-Infection MMP-9 SHAM 3 9 Days Post-Infection 30 108 Figure 2.9: Immunoblotting analysis of TIMPs in CVB3-infected hearts. Western blotting was performed for detection of TIMPs in C V B 3 - and sham-infected mouse hearts at 3 ,9 , and 30 days pi . (A) Representative blots for each protein are shown. (B) A l l o f the blots were normalized to G A P D H and the densitometric graphs are shown as a ratio o f infected to sham (fold change over sham) where sham is equal to one (mean±SD, N=5). TIMP-3 and TIMP-4 were decreased at 9 days pi, the timepoint at which M M P s were upregulated. * /XO.0001 , # /X0.005, % /K0.02, ¥p<0.05. Molecular Weights 30kDa 21kDa 23kDa 23kDa 40kDa Days Post-Infection Sham TIMP-1 TIMP-2 TIMP-3 TIMP-4 G A P D H B TIMP-1 1.4 U 1.0 0.6 O 0.2 fe 0 g 1 4 O 0.2 SHAM 30 i J J A l • • • TIMP-2 S H A M 30 D A Y S P O S T - I N F E C T I O N TIMP-3 S H A M 1.6 u fi 0.6 -J O 0.2 0 DAYS P O S T - I N F E C T I O N TIMP-4 • i n S H A M 30 D A Y S P O S T - I N F E C T I O N DAYS POST-INFECTION 109 2.4 Conclusions and Discussion In this study, I observed increases in M M P - 2 , M M P - 9 , and M M P - 1 2 expression at the transcript and protein levels, as well as in gelatinase activities during acute myocarditis. The cell surface collagenase M T 1 - M M P was also increased while the neutrophil collagenase M M P - 8 was decreased. Their endogenous inhibitors were also differentially regulated: TIMP-1 and TIMP-2 remained constant throughout the infection, while TIMP-3 and TIMP-4 were decreased. The progression from myocarditis to D C M is well documented in both humans and animal models [11,12], although the exact mechanisms have not been elucidated. Virus cleavage o f host structural proteins [13] and cardiac matrical alterations [14] have been proposed to contribute to end-stage disease. M M P - 2 and M M P - 9 have been implicated in cardiac remodeling, such as in human D C M [15] and myocardial infarction (MI) [16], and M M P - 1 2 has also been suggested to play a major role in inflammatory diseases [17]. The role o f collagenases are not well studied in the realm of cardiac diseases but they have been shown to be involved in immune regulation and cellular migration [1,9]. The host immune responses, especially cytokines, are l ikely the major regulators o f M M P s . M M P - 9 expression and activation were increased at 9 days p i , corresponding to previous results [18,19]. Co-localization of M M P - 9 positivity with CD45-expressing cells suggests that infiltrating immune cells are major producers. M M P - 9 has been reported to be secreted by most immune cells in order to aid matrix degradation during migration [20]. Immune cells secrete pro-inflammatory cytokines such as T N F - a and I L - l p which may trigger further M M P upregulation [21]. M M P - 9 has been suggested to play a role in cytokine processing through cleavage and release o f the active molecules, in particular TGF -P and no interleukins [20]. The fact that this protease was increased predominately during the inflammatory phase suggests that it modulates inflammation and early healing, possibly through cytokine regulation. M M P - 2 , has overlapping yet distinct functions relative to M M P - 9 , as evidenced by the difference in localization of these proteases in the heart. Whereas M M P - 9 was localized to infiltrating immune cells, M M P - 2 was more widely distributed and was found in necrotic foci as well as remote cardiomyocytes. I report here that M M P - 2 gelatinolytic activity and expression were increased, similar to that of M M P - 9 . M M P - 2 has been suggested to play a major role in mediating inflammation. Increased M M P - 2 may influence cytokine regulation, cell-to-cell/matrix contacts, and cell migration, all o f which are necessary during wound healing. Recently, M M P - 2 was shown to have intracellular functions, where M M P - 2 was localized to the sarcomeres and nucleus, possibly resulting in myocyte dysfunction [22]. Even though I did observe M M P - 2 expression within cardiocytes, I did not detect cleavage o f troponins nor intermediate filaments following virus infection (data not shown). However, I cannot rule out other unknown intracellular functions of M M P - 2 , such as in signal transduction and cleavage of other molecules within the cell. The role o f metalloelastase in wound healing is relatively unclear due to less extensive studies on this protease in the heart. This is the first study to my knowledge of the characterization of M M P - 1 2 in the myocardium. Recently, many studies have proposed a crucial role of M M P - 1 2 in angiogenesis because this protease cleaves urokinase-type plasminogen activator receptor (u-PAR) and is able to generate angiostatin by cleaving plasminogen, thereby inhibiting angiogenesis [23,24]. Therefore, on one hand, we have upregulation of the gelatinases, which are potent angiogenic molecules, and on the other i l l hand, we have an increase in M M P - 1 2 expression, which may potentially generate inhibitors of vessel growth. Most interestingly, M M P - 1 2 was observed to co-localize to the E C M and cardiomyocytes and not immune cells, particularly macrophages which are thought to be the major source o f this enzyme. This suggests that metalloelastase may play novel and unique roles in the heart. Further investigations into the source and exact localization o f this enzyme is necessary. The collagenases are the only enzymes capable of degrading the fibrillar collagens into forms that can then be further cleaved by other proteases. The major collagenases in mice are m C o l A , m C o l B , M M P - 8 , M M P - 1 3 , and M T 1 - M M P , since rodents lack the homologue o f human M M P - 1 [25]. O f particular interest was that M M P - 8 decreased, notably at the later stages of the disease, while M T 1 - M M P increased substantially, beginning at 9 days pi . Differing results from previous reports showed that M M P - 8 increased during viral myocarditis at 10 days pi and that administration of carvedilol, a beta-adrenergic blocker, decreased M M P - 8 levels, in turn improving the disease condition [26]. M y studies utilized A / J mice while this study performed experiments in Balb/c animals. It has been shown that the mouse strain is important in determining the progression of myocarditis due to varying virulence factors and major histocompatibility loci between strains that ultimately impact the susceptibility of infection and the immune response [5]. In this study, I examined M M P - 8 expression only after 9 days pi , although it would be interesting to examine this enzyme during the early stages of infection, such as at 1-3 days pi , since many studies have shown that M M P - 8 is an important effector of neutrophils, particularly in cytokine processing and the regulation of immune cells [20,27]. Mesenchymal-derived M M P - 8 levels were decreased at 30 days pi , which suggests that fibrotic processes may be developing 112 during this stage of scar formation and maturation. However, M M P - 1 3 was not substantially altered during any stage in our model, which suggests that this enzyme does not play a specific role in myocarditis at the timepoints investigated. Tfie differences in collagenase expression highlight the fact that although there are certain degrees of redundancy between the enzymes, each individual M M P has unique activities and contributes differently to a stimulus despite similar substrate specificities. Examinations into the localization and source of these collagenases as well as investigations at different timepoints of the infection are necessary to clarify their roles in myocarditis. In addition, zymography measurement is not representative of true activity levels because SDS preparation of proteins and incubation with idealized substrates only identifies relative quantities of certain M M P s . Therefore, further experiments are warranted for identification of total M M P activity, such as in vitro cleavage assays o f different substrates. The collective effect o f the TIMPs may have contributed to the increased activity o f the M M P s , resulting in the influx o f immune cells and matrix remodeling. The four T IMPs can inhibit all the active M M P s with some selective affinity; TIMP-1 does not inhibit the membrane-type M M P s very well and TIMP-2 binds to M M P - 2 virtually irreversibly [28]. Even though TIMP-1 has been implicated to play a crucial role in various heart diseases, such as M I and heart failure [29,30], no significant change in TIMP-1 expression was observed in myocarditic hearts. Thus, TIMP-1 may not play a major role in myocarditis or perhaps the selected timepoints did not reflect the change in TIMP-1 expression. Although TIMP-2 expression did not change following infection, its effects may be still important. T IMP-2 , at low concentrations, and M T 1 - M M P synergize to activate p r o M M P - 2 , while at high concentrations, TIMP-2 can inhibit both proteases. Activation of M M P - 2 requires a 113 multi-step and multi-molecule process. First, the N-terminus of T IMP-2 binds to a cell-membrane bound M T 1 - M M P and the C-terminus of the TIMP-2 binds to a p r o M M P - 2 . Primary cleavage of the tethered p roMMP-2 into an intermediate form by an adjacent, uninhibited M T 1 - M M P is followed by final cleavage and activation by an adjacent active M M P - 2 . Corresponding increases in both M M P - 2 and M T 1 - M M P protein expressions in our model suggests that this dynamic may favour activation of M M P - 2 since excess M M P s w i l l diminish the availability of free TIMP-2 , leaving subinhibitory amounts of T IMP-2 to complex with M T 1 - M M P to activate M M P - 2 . Interestingly, both TIMP-3 and TIMP-4 were substantially downregulated during the acute phase of infection. This change may be due to alteration of the cytokine mil ieu and other biological factors that are major modulators of TDMP expression, and may reflect greater roles of these proteins in viral heart disease [28]. The localization and activity levels, which can be assayed using reverse zymography, of the TIMPs w i l l provide further insight into their roles in myocarditis. There are also other MMP-inhibi t ing molecules, such as reversion-inducing cysteine-rich protein with Kazal motifs ( R E C K ) and low density lipoprotein receptor-related protein, which I have not evaluated [31]. In this study, the protein expression and activity levels of M M P s in mouse hearts following C V B 3 infection showed considerable differences and the ultimate result was cardiac remodeling. The picrosirius red-stained hearts showed considerable perivascular, interstitial, and replacement fibrosis at the later timepoints of my study (Figure 2.3), and I also observed substantial loss in ventricular function by echocardiography (data shown in Chapter 6). The last timepoint is 30 days pi , but this is not equivalent to end-stage D C M in human studies. Therefore, further investigation into longer timepoints after infection as well 114 as specific inhibition o f these M M P s w i l l enable us to better understand the role and mechanisms o f these M M P s in cardiac dilation and dysfunction. In conclusion, regulation of M M P - 2 , -9, and -12 in myocarditic hearts is a result o f both differential gene expression and inhibition by TIMPs. These proteases not only function in matrix degradation, but may also be major regulators of cytokines, immune response, angiogenesis, and wound healing, contributing to myocardial injury and remodeling during the progression o f myocarditis. Further elucidation of their mechanisms using transgenic animals and in vitro studies are necessary to dissect out their roles in myocarditis. 115 2.5 References 1. Sternlicht M D , Werb Z . How matrix metalloproteinases regulate cell behavior. Annu Rev Ce l l Dev B i o l 2001;17:463-516. 2. 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Yanagawa B , Spiller O B , Choy J, Luo H , Cheung P, Zhang H M , et al. Coxsackievirus B3-associated myocardial pathology and viral load reduced by recombinant soluble human decay-accelerating factor in mice. Lab Invest 2003;83:75-85. 7. Anderson D R , Wilson JE, Carthy C M , Yang D , Kandolf R, McManus B M . Direct interactions of coxsackievirus B3 with immune cells in the splenic compartment o f mice susceptible or resistant to myocarditis. J V i r o l 1996;70:4632-4645. 8. Tyagi SC, Matsubara L , Weber K T . Direct extraction and estimation o f collagenase(s) activity by zymography in microquantities of rat myocardium and uterus. C l i n Biochem 1993;26:191-198. 9. Quiding-Jarbrink M , Smith D A , Bancroft G J . Production of matrix metalloproteinases in response to mycobacterial infection. Infect Immun 2001;69:5661-5670. 10. Nagase H , Brew K . Designing T I M P (tissue inhibitor of metalloproteinases) variants that are selective metalloproteinase inhibitors. 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Gelatinase B / M M P - 9 and neutrophil collagenase/MMP-8 process the chemokines human G C P - 2 / C X C L 6 , E N A - 7 8 / C X C L 5 and mouse G C P - 2 / L I X and modulate their physiological activities. Eur J Biochem 2003;270:3739-3749. 21. Vreugdenhil G R , Wijnands P G , Netea M G , van der Meer JW, Melchers W J , Galama J M . Enterovirus-induced production of pro-inflammatory and T-helper cytokines by human leukocytes. Cytokine 2000;12:1793-1796. 22. K w a n J A , Schulze C J , Wang W , Leon H , Sariahmetoglu M , Sung M , et al. Matrix metalloproteinase-2 ( M M P - 2 ) is present in the nucleus of cardiac myocytes and is capable of cleaving poly (ADP-ribose) polymerase (PARP) in vitro. F A S E B J 2004;18:690-692. 23. Cornelius L A , Nehring L C , Harding E , Bolanowski M , Welgus H G , Kobayashi D K , et al. Matrix metalloproteinases generate angiostafin: effects on neovascularization. J Immunol 1998;161:6845-6852. 117 24. 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Carboxyterminal cleavage of the chemokines M I G and IP-10 by gelatinase B and neutrophil collagenase. Biochem Biophys Res Commun 2003;310:889-896. 28. Bode W , Fernandez-Catalan C, Grams F, Gomis-Ruth F X , Nagase H , Tschesche H , et al. Insights into M M P - T I M P interactions. A n n N Y Acad Sci 1999;878:73-91. 29. Jayasankar V , Woo Y J , Bish L T , Pirol l i TJ , Berry M F , Burdick J, et al. Inhibition o f matrix metalloproteinase activity by TIMP-1 gene transfer effectively treats ischemic cardiomyopathy. Circulation 2004;110:11180-6. 30. Gaertner R, Jacob M P , Prunier F, Angles-Cano E , Mercadier JJ, Miche l JB . The plasminogen-MMP system is more activated in the scar than in viable myocardium 3 months post-MI in the rat. J M o l Cel l Cardiol 2005;38:193-204. 31. Baker A H , Edwards D R , Murphy G . Metalloproteinase inhibitors: biological actions and therapeutic opportunities. J Cel l Sci 2002;115:3719-3727. 118 CHAPTER III: ABLATION OF MMP-12 IN CVB3-INFECTED MICE INCREASES SEVERITY OF MYOCARDITIS, HEPATITIS, AND PANCREATIS 3.1 Rationale Previous studies performed in our laboratory using an Affymetrix microarray platform found increased expression of M M P - 1 2 or metalloelastase in CVB3-infected mouse hearts, the expression level of which exceeds all other investigated M M P s (please refer to Chapter II, Figure 2.1). In my study detailed in Chapter II, I made novel observations that showed an upregulation of M M P - 1 2 in CVB3-infected mouse hearts, beginning from the early to the chronic phases o f the infection, which appeared to co-localize to cardiomyocytes rather than immune cells. M M P - 1 2 is initially synthesized as a 54 k D a proenzyme that is processed into a 45 k D a intermediate form and then finally cleaved into a 22 kDa active enzyme, whereby the hemopexin domain is lost and only the catalytic domain remains (refer to Chapter I, Figure 1.8 for structural representation). Reports have shown that M M P - 1 2 , like other M M P s , are regulated predominantly at the transcriptional level and various molecules can induce this enzyme, notably statins, IFN-y, and interleukins such as IL-13 and IL-1 P [1-4]. The primary activators of M M P - 1 2 in an inflammatory setting belong to the plasminogen system. Plasmin and thrombin have been reported to induce release of preformed M M P - 1 2 stores in the cytoplasm and these serine proteases can also increase M M P - 1 2 protein secretion and activation through proteolytic conversion [5]. Trrrombin and plasmin can both bind to A version of this chapter will be submitted for publication. Cheung C, Marchant D, Luo H, Yanagawa B, Zhang J, Yuan J, Rahmani M, Walinski H, Walker E, McManus B. MMP-12 deficiency in coxsackievirus-infected mice exacerbates myocarditis, hepatitis, and pancreatis. 119 proteinase-activated receptor-1 (PAR-1) , a G protein-mediated receptor, which induces downstream signalling, possibly through protein kinase C and M A P K family members, to increase protein expression and mobilize intracellular M M P - 1 2 stores for secretion of this enzyme from macrophages [6]. Extensive studies have been performed on M M P - 1 2 in the context of lung disorders, tumour development, and atherosclerosis but very few have investigated the role o f this enzyme in myocardial diseases [7-9]. Previously, reports have shown M M P - 1 2 synthesis in macrophages, chondrocytes, epithelial cells (bronchial, corneal), dermal fibroblasts, and tumour cells [10-14]. These investigations have mainly focused on the roles of M M P - 1 2 in elastinolysis and angiogenesis. M M P - 1 2 has also been reported to facilitate the migration o f monocytes/macrophages to areas of injury. In order to understand the role of M M P - 1 2 in myocarditis, I performed experiments using genetically modified mice deficient in M M P - 1 2 and infected them with coxsackievirus. 3.2 Materials and Methods 3.2.1 In Vitro Study 3.2.1.1 Ce l l Line The HL-1 cell line, generously provided by Dr. Wi l l i am C . Claycomb (Louisiana State University Health Science Center, Baton Rouge, L A ) , was established from mouse atrial cardiomyocyte tumours [15]. These cells proliferate in culture and can be serially passaged, with the ability to retain the differentiated cardiomyocyte phenotype, and the ability to contract. H L - 1 cells were grown in Claycomb medium ( JRH Biosciences, Lenexa, K S ) 120 containing 10% F B S (JRH), 0.1 mmol/L norepinephrine (Sigma-Aldrich), and 2 m M L -glutamine (Sigma-Aldrich). 3.2.1.2 Virus Infection and Detection C V B 3 stocks (Gauntt strain) were propagated by passaging in H e L a cells and the viral titer was measured by agar overlay plaque assay, as described in Chapter II and in previous reports [16]. H L - 1 cells were infected at a multiplicity of infection (MOI) of 100, defined as infection o f 100 P F U o f virus for every cell, with C V B 3 or D M E M alone for 1 hour, washed twice with P B S , and then incubated in fresh Claycomb medium containing 10% F B S at 37°C, as previously described [17]. Cel l lysates and conditioned media were collected and harvested for nucleic acid and protein extractions at 4, 8, 10, and 12 hours pi . CVB3-infected HL-1 cells were also examined for morphological changes by phase-contrast microscopy at 200X magnification at each timepoint and images were taken with a N ikon inverted microscope and Spot digital camera. 3.2.1.3 Nucleic A c i d Extraction and P C R Total mouse R N A was harvested and P C R for M M P - 1 2 was performed as described previously (refer to Chapter II, page 92) [18]. The sequence of the forward C V B 3 primer is 5' G C C T G T G G G T T G A T C C C A C 3' and the reverse C V B 3 primer is 5' G G A A C C G A C T A C T T T G G G T G T C C G T G T T T C 3' [19]. The same P C R condition was used for both M M P - 1 2 and C V B 3 and is listed in Table 2.1, Chapter II, page 92. I performed assays in triplicate at each timepoint. 121 3.2.1.4 Protein Extraction and Western Blot The infected cell fraction was lysed using NP-40 buffer and centrifuged to eliminate the cell debris. Conditioned media was collected and assayed without prior processing. Total protein (20pLg) was electrophoresed in 10% polyacrylamide gels, transferred onto nitrocellulose membranes (Amersham), and probed for M M P - 1 2 , using an antibody from B I O M O L (Plymouth meeting, P A ) at a dilution of 1/1000, and the enterovirus capsid protein VP-1 (Dako), at 1/1500 dilution. Please refer to Chapter II page 94 for further details on the immunoblotting assay. 3.2.2 In Vivo Study 3.2.2.1 Animals and Virus Infection M M P 1 2 K O and their W T counterpart, C57/B16, mice were purchased from Jackson's Laboratory (Bar Harbor, M A , U S A ) . Homozygous knockout mice were bred at St. Paul's Hospital in the Genetically Engineered Mouse facility. Five week-old adolescent males from both genotypes were infected intraperitoneally with 10 5 P F U of C V B 3 or P B S . Infected mice were sacrificed at 3 days pi due to the severe moribund condition of the K O mice. In total, 19 W T C57/B16 and twelve M M P 1 2 K O mice were used in this study. A l l animals were weighed daily following infection, and their vital signs and morbidity states were recorded. A l l animal procedures were in accordance with the Animal Care Committee, U B C . 3.2.2.2 Histological Assessment Various organs, including pancreas, liver, mid-ventricular myocardium, and spleen were collected, fixed in 10% neutral-buffered formalin, and embedded, in paraffin. The tissues were sectioned at 4-5p^m, and processed for H & E staining to detect areas of organ damage and inflammatory infiltration, Movat 's pentachrome for matrix composition, and 122 picrosirius red staining for structural integrity of collagen. The H & E stained sections were graded in a blinded fashion by a pathologist on a scale o f 1 (minimal damage) to 5 (maximum damage), as described in Chapter II page 90. Images were all captured using a Nikon inverted microscope and Spot digital camera. A l l animals were included in these assessments. 3.2.2.3 Virus Detection and Plaque Assay The basal portion of the heart tissue was flash-frozen in liquid nitrogen, homogenized in D M E M , and subjected to plaque assay for presence of virus, as described in Chapter II, page 87. Eight to twelve animals were used for this assay. 3.2.2.4 Immunohistochemistry Paraffin-embedded heart sections were immunolabeled for the presence and localization of V P 1 . Briefly, slides were dewaxed, rehydrated, heated in 6% urea for 10 minutes by microwave, and incubated overnight at room temperature using an anti-VP 1 antibody (Novocastra/Vision BioSystems, Norwell , M A ) at 1/75 dilution in serum-free protein block (Dako), and detected using the ABComplex amplification system with D A B as substrate. Images were all captured using a Nikon inverted microscope and Spot digital camera. Eight to twelve animals were used for this assay. 3.2.2.5 T U N E L Staining Ce l l death and apoptosis in the organs were assessed using an ApopTag® Plus Peroxidase In Situ Apoptosis K i t (Chemicon) according to manufacturer's protocol. Briefly, formalin-fixed and paraffin-embedded tissue sections were rehydrated, treated with proteinase K (20pig/mL) for 15 minutes at room temperature, incubated with 3% hydrogen peroxide to quench endogenous peroxidase, treated with TdT enzyme, incubated with anti-digoxigenin 123 conjugate, and visualized using D A B colour substrate and counterstained with methyl green. Eight to twelve animals were used for this assay. 3.2.2.6 Data and Statistical Analyses The N I H ImageJ 1.3 l v program (http://rsb.info.nih.gov/ij/) was utilized to quantitate Western blot and zymography band intensities. The data is presented as mean ± SD in the text and graphs. Pairwise comparisons between virus-infected W T and M M P 1 2 K O mice were conducted using two-tailed, unpaired Student's t-test or the Wilcoxon non-parametric test with the J M P (Cary, N C ) software program, and differences were considered significant at p<0.05. 3.3 Results 3.3 . 1 In Vitro Study 3.3.1.1 Cultured Murine Cardiomyocytes HL-1 cells are cultured atrial cardiomyocytes capable of contraction in culture and retention o f the cardiac phenotype even after multiple passaging cycles. Virus-infected H L - 1 cells first began to show signs of cytopathic effects, such as detachment from plate substratum and cellular condensation, at 4 to 5 hours pi and cell death usually occurs at 8 to 9 hours pi (Figure 3.1), similar to the well-established in vitro model using HeLa cells [20]. B y 24 hours pi , most of the cells were lysed. 124 Figure 3 .1: Coxsackievirus infection of H L - 1 cells. Shown here are phase-contrast micrographs o f cultured HL-1 cells after C V B 3 infection. (A) Sham, (B) 1 hour, (C) 4 hours, (D) 9 hours, (E) 12 hours, and (F) 24 hours following coxsackievirus infection. Images were taken at 200X magnification. 125 3.3.1.2 Transcriptional and Translational Expression of M M P - 1 2 in HL-1 H L - 1 cells were infected with C V B 3 and their nucleic acids were harvested at 4, 8, and 12 hours p i , which are reflective of the different stages of the virus infection, as described in Chapter I, page 15. M M P - 1 2 is produced constitutively at low levels even in sham cardiomyocytes but transcription of this enzyme peaked at 4 hours pi (p<0.05), as shown in Figure 3.2A. Figure 3.2B shows the densitometric analysis of M M P - 1 2 bands. P C R for the virus confirmed the presence of viral genome in the infected cells. Figure 3.2C shows the protein expression of M M P - 1 2 in the conditioned media and cell lysates from virus-infected HL-1 cells. The pro form of M M P - 1 2 , which is observed as a 60 k D a band on a Western blot, was detected in the media, as expected since M M P - 1 2 is a secreted protein and does not reside in intracellular compartments or at the cell surface. A very faint band could also be detected at 50 kDa, which may be the active form o f M M P - 1 2 . The discrepancy between the amount of M M P - 1 2 m R N A and protein can be explained by the possibility that the half-life of this enzyme is longer than our experimental time. The diminishment of transcription after 8 hours pi may be due to excessive cell death o f the infected cells after this timepoint. V P 1 confirmed the presence of viral protein within the infected cells but not in the conditioned media and as shown previously, VP1 could be detected only after 4 hours pi possibly due to a lag between transcription and translation in the infected cells or because the level of VP1 protein was below detection by Western blotting [20]. 126 Figure 3 . 2 : Expression of MMP-12 and C V B 3 in infected HL-1 cells. (A) Transcriptional expression of M M P - 1 2 (top) and C V B 3 (middle) at 4 , 8, and 12 hours pi . (B) Densitometric comparisons (mean±SD) of the P C R bands between infected and sham samples show that expression of M M P - 1 2 peaked at 4 hours pi . Results are expressed as intensity in arbitrary units. (C) Translational expression of M M P - 1 2 and VP1 in conditioned media and cell lysates of CVB3-infected HL-1 cells. M M P - 1 2 was secreted as a proform into the media even in sham-infected cultures and the expression was increased following coxsackievirus infection. A representative blot is shown and an individual sample was run per lane. Experiments were performed in triplicates. ¥p<0.05. Sh 8 12 • m-m mm « w * # • . m mm mm pm M • - n**a • M a f faaap #>»•* ***•> « hrs p i MMP-12 CVB3 18s rRNA B OS fl 0 ) 0 Sham 4 8 Hours Post-Infection Media Cell Lysates 60kDa 50kDa 32kDa 36kDa 10 12 hrs p i MMP-12 GAPDH 127 3.3.2 In Vivo Study 3.3.2.1 Morbidity and Histological Examination Sham-infected M M P 12KO and W T mice did not exhibit any histological, growth, and physical abnormalities (data not shown). Following infection, the M M P 1 2 K O mice appeared to suffer more severe disease as reflected by an increase in mortality (33% in M M P 1 2 K O vs. 0% in W T , /><0.05) and morbidity, as measured by decrease in body weight (16.7±1.8% in M M P 1 2 K O vs. 4 .4±4.6% in W T , /?=O.002), represented in Figure 3.3A. In the infected M M P 1 2 K O mouse hearts, there appeared to be increased cytolysis and cardiomyocytes undergoing cell death, as indicated by vacuolization and contraction banding o f cells (Figure 3.3B, arrow). In the pancreas of all M M P 1 2 K O mice, the exocrine tissue, consisting of acinar cells, was completely destroyed (Figure 3.3D). However, the endocrine glands, including the islets of Langerhans and pancreatic ducts (long arrows), were uninfected and remained viable throughout the infection, as previously described [21]. In the W T pancreas, many viable acinar cells were still detected, as indicated by the short arrows in Figure 3.3E. In the case o f the M M P 1 2 K O livers, there was widespread hepatocytolysis as well as moderate immune infiltration, resulting in loss of structural integrity (Figure 3.IF). Apoptotic hepatocytes and immune cells were also detected in the W T livers (Figure 3.3G), albeit at a much lower frequency. The cause o f mortality in the M M P 1 2 K O mice was probably hepatitis, extensive liver injury, and dysfunction. The spleens of the M M P 1 2 K O mice were also morphologically altered as compared to W T mice (Figures3.3H and 3.31). The M M P 1 2 K O spleens were more atrophic, there were much larger and more frequent germinal centers, and within the follicles, there was overt widespread apoptosis. Megakaryocytes and giant cells were also occasionally seen in the K O spleens. 128 Pathological scoring of H&E-stained tissue confirmed that the M M P 1 2 K O suffered more severe damage in the heart (2.25±1.2 in M M P 1 2 K O vs. 0.6±0.6 in W T , ^=0.005) pancreas (5.0±0.0 in M M P 1 2 K O vs. 4.7±0.4 in W T , /?=0.001), liver (3.6±1.1 in M M P 1 2 K O vs. 1.7±0.8 in W T , p=0.02), and spleen (4.0±0.3 in M M P 1 2 K O vs. 1.6±0.6 in W T , p«0.00\) as summarized in Figure 3.3J. Figure 3.3: Morbidity and mortality assessment of CVB3-infected M M P 1 2 K O and WT mice at 3 days pi. (A) M M P 1 2 K O mice experienced increased mortality (33%) as compared to W T (0%) and an increase in morbidity, as measured by decrease in body weight. (B-I) CVB3-infected heart, pancreas, liver, and spleen from both genotypes were stained with H & E and scored for injury and inflammation. (B) M M P 1 2 K O myocardium experienced more injury and cells undergoing cell death, as evidenced by contraction banding and vacuolization (arrow), as compared to (C) W T mice. (D) Exocrine pancreas in M M P 1 2 K O mice was completely destroyed, saving the islets of Langerhans [long arrows], while viable acinar cells were detected in the (E) W T mice [short arrows]. (F) There was widespread hepatocytolysis and immune infiltration in M M P 1 2 K O mouse livers while (F) W T mice experienced a lesser degree o f injury. Increased apoptosis was detected in the germinal centers o f (G) M M P 1 2 K O as compared to (H) W T mice. (J) Semi-quantitative histological scores comparing M M P 1 2 K O and W T are displayed as mean±SD. The bars within the images denote 50|am. Each figure is a representative from an individual mouse. * /K0 . 0001 , # / K 0 . 0 0 5 , % p<0.02, ¥ p<0.05. 1 2 9 MMP12KO WT s» MMP12KO WT Genotypes Genotypes MMP12KO WT Genotypes 130 3.3.2.2 Matrix Composition and Fibrosis Since elastin is a major substrate of M M P - 1 2 , we used Movat 's pentachrome stain to determine the composition of matrix, of which black stains elastic fibers, green is proteoglycans, and yellow denotes collagen. However, since collagen is difficult to detect with this stain, I w i l l only discuss elastic fibers and proteoglycans here. A s shown by the Movat 's stain, the hearts did not experience any substantial alteration in matrix composition (Figures 3.4A and 3.4B). There did not appear to be any degradation or accumulation o f elastin and proteoglycans within the myocardium of both M M P 1 2 K O and W T mice. Elastic fibers in the myocardium of both genotypes were maintained and were detected predominantly lining the vessel walls (arrows). Rare accumulation of proteoglycans was detected in the hearts. In the pancreas, despite a significant difference between the histological grades o f M M P 1 2 K O and W T mice, the matrix composition did not appear altered between the two genotypes. There was massive destruction of the exocrine pancreas in both genotypes and abundant accumulation o f proteoglycans were found surrounding dying or dead cells as well as around vessels and ducts (arrows), as shown in Figures 3.4C and 3.4D. There were little or no alterations o f the elastic fibers, where they predominantly line vessel walls. In the liver, the majority of matrix material consisted of collagens while elastic fibers were found mostly within vessel walls and proteoglycans were scarce in this organ. Despite the overt histological changes, as evidenced by H & E stains, in the M M P 1 2 K O mice as compared to the W T , little or no substantial matrical changes were detected in this organ (Figures 3.4E and 3.4F). 131 Lastly, I investigated the composition of elastin and proteoglycans in spleen. Similar to the heart and liver, proteoglycans were rarely detected in this organ in both genotypes but elastin and collagen were abundant, frequently detected around the trabecula vessels (arrows), as shown in Figures 3.4G and 3.4H. However, there did not appear to be any substantial difference in the amount and orientation o f these fibers between M M P 1 2 K O and W T mice. Figure 3.4: Mova t ' s staining of CVB3-infected M M P 1 2 K O and W T mice. Representative micrographs of Movat 's staining of (A) M M P 1 2 K O and (B) W T myocardium; (C) M M P 1 2 K O and (D) W T pancreas; (E) M M P 1 2 K O and (F) W T livers; and (G) M M P 1 2 K O and (H) W T spleens. The purple stains cytoplasm, black dots are nuclei, black stain is for elastic fibers, blue/green stains proteoglycans, and yellow denotes collagen. Each figure is a representative from an individual mouse. The bars within the images denote 50pim. To investigate the composition of collagens, o f which the fibrillar types I and III are the most abundant forms, I used picrosirius red stain, where muscle is stained yellow and collagen is stained red. The collagen matrix forms very fine tethers between the cardiomyocytes as well as between myocytes and vessels, termed endomysium, while thicker 132 collagen filaments, called perimysium, surround bundles of muscle fibers [22]. The collagen structure within the heart was not substantially perturbed at 3 days p i , as previously described [18]. There was also no significant difference in collagen accumulation between M M P 1 2 K O and W T mice (Figures 3.5A and 3.5B). Using light microscopy, I was able to detect the thick perivascular collagen matrix (short arrows) and the perimysial tethers (long arrows) but not the fine struts and weaves of the endomysium. I did not detect any apparent disorganization or dissolution o f the collagen matrix in the heart o f both genotypes and there was no significant difference in the collagen organization between M M P 1 2 K O and W T myocardium. In the pancreas, there was massive destruction and cell death and this resulted in substantial alteration in the collagen matrix. There was marked increase in fibrosis and the collagen matrix organization in these areas was disoriented and disarrayed. The collagen bundles did not appear to form tight fibers but were diffusely arranged (arrows), as shown in Figures 3.5C and 3.5B. This corresponded to the Movat 's stains where accumulation o f proteoglycans was detected in the infected pancreas, which may result in abnormal collagen organization. However, despite a deficit in M M P - 1 2 , the K O pancreas were not significantly different as compared to W T . Upon investigation of the livers, substantial differences between M M P 1 2 K O and W T mice were observed (Figures 3.5E and 3.5F). The amount of collagen accumulation was not affected between M M P 1 2 K O and W T livers but I detected altered collagen structures in the K O mice. In both strains of mice, the arrangement of collagen around the vessels appeared normal and did not exhibit fibrotic tendencies. However, in the W T mice, the intra-hepatocyte collagen network (arrows) appeared intact but in the M M P 1 2 K O mice, there was 133 complete dissolution of this network. This corresponds to the observed massive hepatocytolysis in the M M P 1 2 K O mice whereas the W T livers experienced only mild injury. Lastly, I investigated the collagen matrix in the spleens of the infected mice. I detected intact trabecula vessels and normal collagen architecture in both genotypes (Figures 3.5G and 3.5H). Despite experiencing more injury, there appeared to be no significant difference between the infected M M P 1 2 K O and W T spleens. Figure 3.5: Picrosir ius red staining of CVB3-infected M M P 1 2 K O and W T mice. Fibrosis and collagen architecture were assessed by picrosirius red staining using light microscopy. Representative stained images of (A) M M P 1 2 K O and (B) W T myocardium; (C) M M P 1 2 K O and (D) W T pancreas; (E) M M P 1 2 K O and (F) W T livers; and (G) M M P 1 2 K O and (H) W T spleens are shown. Little or no substantial alterations in collagen amount and organization were detected in the myocardium, pancreas, and spleen between M M P 1 2 K O and W T mice. Although there was no difference in collagen accumulation between livers of M M P 1 2 K O and W T mice, the collagen matrix appeared disorganized and disarrayed in the M M P 1 2 K O mice. Ye l low stain denotes muscle or cytoplasm and the red stains for collagen. Each figure is a representative from an individual mouse. The bars within the images denote 50pjn. A C . / . ' £ •rs . . . . . . A -. . . 1 * ^ : p G . • ; V y] • • :'-:'::.:'.:t \ K -B ... v .. ...... V^B-Vfc ; D - WB&fi* 1 "-. •• , - . •» . 134 3.3.2.3 Virus Detection and Quantitation Plaque assay was performed to determine the presence and amount of virus within CVB3-infected hearts (Figure 3.6A). M M P 1 2 K O had increased viral load, more than 1.5 unit on a loglO scale (p=0.007), within the myocardium following C V B 3 infection as compared to W T mice. This was confirmed using immunohistochemistry for the enterovirus capsid protein V P 1 . While very little immunoreactivity for VP1 was detected in the W T mouse hearts, I observed diffusely infected cardiomyocytes in the left ventricles o f the M M P 1 2 K O mice (Figures 3.6B and 3.6C, arrows). The amount of infection in the pancreas (Figures 3.6E and 3.6F) and liver (Figures 3.6H and 3.61) was also assessed via immunohistochemical staining for V P 1 , which showed increased expression of this protein in M M P 1 2 K O as compared to W T within these organs. A s expected, the endocrine system in the pancreas was not infected, for no immunoreactivity was detected within these cells (short arrows). The M M P 1 2 K O livers had widespread infection while in the W T , there were sparsely infected hepatocytes (arrows). This correlates with the histological stains and supports the cause of death by hepatic dysfunction. In the spleen, very little VP1 immunoreactivity was observed in the W T mice and diffusely stained cells were detected in the red pulp region o f the M M P 1 2 K O spleens (arrows), as shown in Figures 3.6K and 3.6L. Very few V P 1-stained cells were detected within the germinal centers. Figures 3.6D, 3.6G, 3.6J, and 3 .6M are the negative controls for the immunohistochemistry and confirmed specificity of the antibody for VP-1 as no immunoreactivity was observed in these sections. 135 Figure 3.6: Assessment of virus infection i n M M P 1 2 K O and W T mice. (A) Standard agar plaque assay was performed to determine the amount o f viral particles in the infected myocardium, calculated as # PFU/microgram of heart tissue, and graphed on a loglO scale. (B) Representative micrographs which show immunostaining for VP1 in (B) M M P 1 2 K O and (C) W T myocardium; (E) M M P 1 2 K O and (F) W T pancreas; (H) M M P 1 2 K O and (I) W T livers; and (K) M M P 1 2 K O and (L) W T livers are shown. Negative controls for immunohistochemical staining are shown in (D) myocardium, (G) pancreas, (J) liver, and (M) spleen. Immunoreactivity is shown in brown and blue denotes nuclei. Each figure is a representative from an individual mouse. Scale bars denote 50fim. % p<0.02 A '3D o (J 14 -I o 3 12 SB ga 'rZ. — 10 t 8 eg <u 6 S 01 4 a 2 -0 Cu MMP12KO WT Genotypes , ,- * v ; '•'•,-'"'.'* ' . "J.-; , : : • • : : • ' } • ' ' ' ' ^ H .... • c V:;?Z.); l .'-';--'r v l i Bars=50um 136 3.3.2.4 Apoptosis I next investigated the frequency of cell death by apoptosis in the virus-infected M M P -12KO and W T mice. T U N E L staining was performed and is presented in Figure 3.7. In the myocardium, apoptosis of cardiomyocytes was extremely rare in both genotypes and not all o f the investigated portions of the myocardium contained apoptotic cells. Figures 3.7A and 3.7B show TUNEL-stained cells that were possibly undergoing apoptosis (arrows). In the pancreas, even though all o f the exocrine glands were destroyed in the M M P 1 2 K O mice, T U N E L staining was not detected in the whole organ because the endocrine components were not affected by the infection and some of the acinar cells had possibly undergone necrosis instead of apoptosis (Figure 3.7C). B y visual inspection, there appears to be more apoptotic cells within the M M P 1 2 K O pancreas than in the W T ones due to the increased presence of viable acinar cells in the W T pancreas (Figure 3.7D). In the liver, I detected widespread apoptotic hepatocytes in the M M P 1 2 K O mice while W T mouse livers also experienced cell loss, albeit at a much lower frequency (Figures 3.7E and 3.7F). The increased hepatocytolysis probably contributed to the loss of structural integrity of this organ and led to liver dysfunction. Within the M M P 1 2 K O spleen, I observed a similar pattern as the liver, where widespread apoptosis within the germinal centers as well as occasional apoptotic bodies in the red pulp area were detected. In the W T mice, the spleens had sparse apoptotic cells in the germinal centers as well as infrequent apoptotic cells in the red pulp area, as shown in Figures 3.7G and 3.7H. This correlates with the histological and the VP1 immunohistochemistry results. 137 Figure 3.7: Apoptosis analysis of CVB3-infected M M P 1 2 K O and W T mice. T U N E L staining was performed to detect apoptosis in (A) M M P 1 2 K 0 and (B) W T myocardium; (C) M M P 1 2 K O and (D) W T pancreas; (E) M M P 1 2 K O and (F) W T livers; and (G) M M P 1 2 K O and (H) W T spleens. Immunoreactivity is shown in brown while nuclei are stained green. Each figure is a representative from an individual mouse. Scale bars denote 50|im. G ' - , ; ' . -B D *. - . -0 F ' ' • . • • ' J V:-: : H - — » '.. • - : > • !r**"** • 3.4 Conclusions and Discussion In this study, I investigated the role of M M P - 1 2 using in vitro and m vivo models. I infected HL-1 cells and determined the transcriptional and translational expression o f M M P -12 following coxsackievirus infection. I detected increased expression of both transcript and enzyme as early as 4 hours pi , when cytopathic effects and cell death had not yet begun. To further investigate the importance of this protein, I used a well-established coxsackievirus-induced myocarditis murine model utilizing genetically modified mice deficient in M M P - 1 2 . M M P 1 2 K O and their corresponding W T s were infected with C V B 3 and then I compared tissue injury, matrix composition, viral load, and cell death between these mice. M M P 1 2 K O 138 mice experienced increased mortality and morbidity as well as more severe cardiac, pancreatic, hepatic, and splenic injury, increased viral abundance, and increased cell death as compared to W T . However, the elastin matrix was not overtly altered between the two genotypes and excessive accumulation of collagen was only observed within the K O pancreas while deterioration of the collagen organization was detected in the M M P 1 2 K O livers. The primary cause of death in the M M P 1 2 K O animals appears to be hepatitis and structural disintegration leading to liver dysfunction. Thus, this study suggests that M M P - 1 2 plays a major protective role during the acute phase of viral infection. I observed an increase in M M P - 1 2 expression in CVB3-infected HL-1 cells as early as 4 hours p i , at which time the host cell defense mechanisms have already been activated and the virus is propagating but cytolysis has not yet occurred [20,23]. Upregulation of M M P - 1 2 as well as a cluster of other M M P s have been reported in other viral infections, notably Theiler murine encephalomyelitis virus ( T M E V ) which also belongs to the Picornaviridae family and mouse hepatitis virus [24,25]. These studies propose that M M P s facilitate the recruitment, migration, and activation of immune cells such as T-lymphocytes into the infected organ and contribute to disease through disruption of the parenchymal E C M . However, immunosuppression in viral encephalitis did not inhibit M M P transcription but in contrast, expression of M M P - 3 , M M P - 1 2 , and TIMP-1 was increased [25]. Further, persistent viral infection, as shown by chronic detection of T M E V genome in the infected organ, was associated with M M P - 1 2 expression in the infected cells. This suggests that virus replication itself is sufficient to induce the expression o f M M P s in the absence of inflammation. Previous studies from our laboratory has demonstrated that C V B 3 upregulates several key signalling pathways, including E R K and p38 pathways, which are known to increase the 139 activity o f AP-1 and c-Jun transcription factors that in turn increase M M P - 1 2 expression . However, no studies thus far have shown that virus can directly increase M M P - 1 2 expression. Our laboratory have shown that the virus regulates the expression of host proteins by manipulating host signalling pathways, such as the extracellular signal-regulated kinase ( E R K ) and stress-activated protein kinase (consisting of cJun N-terminal [JNK] and p38 kinases) transduction pathways, which ultimately activates the AP-1 transcription family [26,27]. The regulation of M M P s is predominately through the AP-1 family of transcription factors, thus this is potentially a mechanism by which coxsackievirus can upregulate M M P - 1 2 expression. Some reports have shown that viruses may mediate M M P expression through cytokine/growth factor mechanisms. For example, IL-1 (3 and IFN-y, both of which are secreted by infected cells early on during virus infection, have been shown to upregulate expression and activation o f M M P - 1 2 [2,4]. Further, it has been shown that IL-13 can induce the expression o f M M P - 1 2 , which in turn can enhance the induction of other M M P s ( M M P - 2 , M M P - 9 , M M P - 1 3 , and M M P - 1 4 ) [3]. M M P - 1 2 is able to degrade a wide variety o f substrates, but elastin and plasminogen have been the most extensively studied. The degradation of elastin is particularly relevant in the lung and vessel wall , since this fibrillar protein maintains the structure and function of these tissues, but elastin has not been shown to play a major role in the heart, liver, pancreas, and spleen. Many investigators have shown a link between upregulation o f M M P - 1 2 and respiratory disorders, such as chronic obstructive pulmonary disease (COPD) and emphysema [28]. Alveolar macrophages have been reported to increase expression and secretion o f M M P - 1 2 upon stimulation by cigarette smoke and in turn, this enzyme can degrade elastin, resulting in disruption of the elastic network leading to structural deformations and generation 140 of elastin fragments that have chemotactic activity leading to P M N and monocyte recruitment to the lung [29]. In the vasculature, infiltrating macrophages secrete abundant amounts of M M P - 1 2 , leading to degradation o f the elastic laminae and rupture of the vessel wall [9]. Using genetically modified mice deficient in M M P - 1 2 , previous reports have shown that loss o f this enzyme preserves the integrity of the elastin network in airway and vessel walls and abates the inflammatory response via a decrease in immune cell migration [7,9]. However, in our model, loss o f M M P - 1 2 in contrast increases the severity o f the infection in multiple organs but the elastin network, as well as collagen and proteoglycan deposition, did not experience significant accumulation nor was there any apparent disorganization as visualized using Movat 's staining (Figure 3.4). Only the collagen matrix within the M M P 1 2 K O liver was severely perturbed, which may have led to structural disintegration and organ dysfunction. M M P - 1 2 has been shown to cleave collagen type IV, found predominately in the basal laminae surrounding individual cells but this enzyme cannot cleave fibrillar collagens. Therefore, in our viral model, deficiency of M M P 12 induced inappropriate degradation of the collagen matrix in the liver, possibly through upregulation of other proteases. In order to clarify this, examination of other M M P s and TIMPs is necessary. M M P - 1 2 has been implicated to play a major role in regulation o f angiogenesis through proteolytic cleavage of plasminogen and generation o f angiostatin, which is a potent inhibitor of angiogenesis [30]. This is especially important in the setting of tumour development and wound healing, since the blood supply is crucial in the migration, proliferation, and nutrient distribution of cells. It is possible that in the M M P 1 2 K O mice infected with C V B 3 , loss of M M P - 1 2 may decrease the amount of angiogenesis, therefore, delaying wound healing. I detected a substantial amount of infiltrating immune cells in the 141 pancreas as well as in the liver, thus confirming normal extravasation of immune cells and proper vasculature into the wound sites. However, I did not specifically investigate the amount of new microvasculature generated in these organs following infection and a decrease in microvasculature may result in hypoxic conditions that are damaging to the tissue. Generation of R O S is a byproduct of hypoxic conditions and R O S can induce the activation o f a variety o f M M P s , such as M M P - 2 and M M P - 9 , which are known to be capable o f degrading fibrillar collagen and regulating immune infiltration. Future investigations into the M M P profile in M M P 1 2 K O mice would reveal whether compensation by other M M P s had occurred. Previous studies have also attributed M M P - 1 2 to recruitment and migration of immune cells to the wound sites. IL-13-induced inflammation was shown to induce M M P - 1 2 expression in alveolar macrophages, which contributed to the recruitment and accumulation of eosinophils and macrophages in the lung [3]. Indeed, direct instillation of recombinant human M M P - 1 2 into mouse airways elicited an intense inflammatory response, characterized by the development of two stages: 1) an acute and intense recruitment o f P M N s , which peaked at 18 hours post-instillation; and 2) induction of IL-6, K C (IL-8), macrophage inflammatory protein-la, and M M P - 9 levels, resulting in late-stage macrophage recruitment [28]. Therefore, M M P - 1 2 appeared to be an early inducer of the innate immune response as well as a delayed inducer of macrophages and lymphocytes. During virus infection in mice, immune infiltration does not enter the myocardium until after 4 days pi but inflammation could be detected in the pancreas and liver within 1-2 days pi [16]. The loss of M M P - 1 2 in C V B 3 infection could result in a downregulation of the innate immune system, possibly by a decrease in recruitment and migration of P M N s into the wound sites. This may result in 142 inefficient viral clearance and virus control, which may aid in virus replication, dissemination, and increased secondary infections of neighbouring cells, leading to a more serious infection. We observed an increase in infiltrating cells in the pancreas and liver of the M M P 1 2 K O mice but we did not specifically investigate the subtypes of these cells and this increase in infiltration could be a delayed response to the elevated severity of the infection due to ineffective primary immune response. Investigations at earlier timepoints (0.5, 1, 2 days pi) may clarify this issue. M M P - 1 2 can also mediate the innate immune response through regulation of cytokines and chemokines. It has previously been shown that M M P - 1 2 can directly activate TNF-oc secreted from macrophages, with subsequent P M N influx and proteolytic matrix breakdown caused by immune cell-derived proteases [7]. T N F - a is a pleiotropic cytokine and a major regulator o f wound healing, inflammation, apoptosis, cellular migration, and cellular proliferation. It has been shown that longterm upregulation o f T N F - a is detrimental due to induction of continual inflammation but short term balanced expression of T N F - a is crucial to repair and maintenance of homeostasis. Further, as described above, IL-13 induction of M M P - 1 2 directly mediates the production of other M M P s , such as M M P - 2 , M M P - 9 , M M P -13, and M M P - 1 4 . This family of enzymes have been implicated in cytokine/chemokines processing, whereby M M P s can activate and/or inactivate bioactive molecules upon stimulation. Therefore, loss of M M P - 1 2 may disrupt the balance of cytokines and chemokines, resulting in uncontrolled viral clearance and a delay in wound healing. M M P - 1 2 can also degrade other molecules, including collagen type I V and laminin (both major constituents of the basal laminae), gelatin (degraded form of fibrillar collagen), fibronectin, vitronectin, and a-1-proteinase inhibitor [28,31]. The inability of M M P 1 2 K O 143 mice to effectively cleave basal laminae substrates may result in damage to both the infected and adjacent cells, since cellular repositioning through modulation o f E C M interactions may be crucial to the survival of the cells. After the loss of cells through virus-mediated cytolysis, repositioning of the remaining viable cells by formation of new cell-cell and cell-matrix contacts is necessary in order to prevent anoikis and to maintain tissue structure. Excessive loss of cell-cell or cell-matrix interactions due to increased protease activities and pericellular proteolysis may result in anoikis, defined as programmed cell death induced by the loss o f these interactions [32]. In this study, I observed increased apoptosis in various organs, including the pancreas, liver, and spleen, in the infected K O mice as compared to controls (Figure 3.7). This contrasting phenomenon could occur in the M M P 1 2 K O mice via the inability o f the remaining viable cells to form proper cell-cell and cell-matrix contacts because M M P s are required for the degradation of the basal laminae and other E C M components in order for the cells to reform cellular contacts [28,31]. It would be interesting to determine i f the apoptotic cells are infected or not by performing in situ hybridization for the C V B 3 genome in conjunction with T U N E L staining. The inability of M M P 1 2 K O mice to properly degrade matrix can also affect wound healing since fibronectin and vitronectin, which are major components of the provisional matrix, are some substrates of M M P - 1 2 . A s discussed earlier in Chapter I, proper organization, resolution, and resorption of the provisional matrix is necessary in order for effective recruitment, migration, and adhesion o f infiltrating cells, such as myofibroblasts. Therefore, in M M P - 1 2 deficient mice, improper provisional matrix development, especially of fibronectin, w i l l hinder the adhesion of myofibroblasts and organization o f the collagen 144 network, leading to loss of proper cell-cell and cell-matrix interactions as well as delayed wound healing. M M P - 1 2 is shown in this study to play a protective role against virus infection in multiple organs during the early phase of the disease (day 3 pi). These results contrast other published studies where the loss of M M P - 1 2 prevented or abated disease progression by limiting the inflammatory response. In coxsackievirus infection, early activation of the immune response is necessary to efficiently clear the virus and to initiate the wound healing response. Despite all the observational results generated in this study, the mechanisms by which M M P - 1 2 operates is still unknown. A s discussed above, more detailed investigations into the matrix composition and organization is needed to determine any changes from loss of M M P - 1 2 . In addition, the cause of apoptosis in the various organs in the M M P 1 2 K O mice requires further investigation to determine i f it is virus-dependent or ECM-mediated. The use of genetically modified mice can generate many observations but much more questions arise from the results, thus in vitro experiments are necessary in order to better define the role of M M P - 1 2 in C V B 3 infection. 145 3.5 References 1. Ar ikan M C , Shapiro SD, Mariani TJ . Induction of macrophage elastase (MMP-12) gene expression by statins. J Cel l Physiol 2005;204:139-145. 2. Wang Z , Zheng T, Zhu Z , Homer RJ , Riese R J , Chapman HA,J r , et al. Interferon gamma induction of pulmonary emphysema in the adult murine lung. J Exp M e d 2000;192:1587-1600. 3. Lanone S, Zheng T, Zhu Z , L i u W , Lee C G , M a B , et al. Overlapping and enzyme-specific contributions of matrix metalloproteinases-9 and -12 in IL-13-induced inflammation and remodeling. J C l i n Invest 2002; 110:463-474. 4. Lappalainen U , Whitsett J A , Wert SE, Tichelaar JW, Bry K . Interleukin-lbeta causes pulmonary inflammation, emphysema, and airway remodeling in the adult murine lung. 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Si X, Luo H, Morgan A, Zhang J , Wong J , Yuan J , et al. Stress-activated protein kinases are involved in coxsackievirus B3 viral progeny release. J Virol 2005;79:13875-13881. 27. Luo H, Yanagawa B, Zhang J , Luo Z, Zhang M, Esfandiarei M, et al. Coxsackievirus B3 replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway. J Virol 2002;76:3365-3373. 28. Lagente V, Manoury B, Nenan S, Le Quement C, Martin-Chouly C, Boichot E. Role of matrix metalloproteinases in the development of airway inflammation and remodeling. Braz J Med Biol Res 2005;38:1521-1530. 29. Houghton AM, Quintero PA, Perkins DL, Kobayashi DK, Kelley DG, Marconcini LA, et al. Elastin fragments drive disease progression in a murine model of emphysema. J Clin Invest 2006; 116:753-759. 30. Cornelius LA, Nehring LC, Harding E, Bolanowski M, Welgus HG, Kobayashi DK, et al. Matrix metalloproteinases generate angiostatin: effects on neovascularization. J Immunol 1998;161:6845-6852. 31. Chakraborti S, Mandal M, Das S, Mandal A, Chakraborti T. Regulation of matrix metalloproteinases: an overview. Mol Cell Biochem 2003;253:269-285. 3 2 . Michel JB . Anoikis in the cardiovascular system: known and unknown extracellular mediators. Arterioscler Thromb Vase Biol 2003;23:2146-2154. 148 CHAPTER IV: MMP-9 DEFICIENCY INCREASES SEVERITY OF VIRAL MYOCARDITIS FOLLOWING CVB3 INFECTION 4.1 Rationale During the inflammatory phase, massive lymphocytic infiltration, including macrophages, T-cells, N K cells, and granulocytes enter the heart to clear the virus [1,2]. Inadequate viral clearance may lead to increased virus-induced cytolysis and virus persistence in the myocardium and chronic myocarditis [3,4]. Ablation of inflammatory cells by using genetically modified mice or antibody hindrance technique in the study of viral myocarditis have generated many interesting results, which suggests that a balance of pro- and anti-inflammatory processes is necessary in order to achieve complete cardiac repair and resolution. MMPs are important regulators of inflammation through their matrix-degrading functions as well as in cytokine processing and cell migration, as shown in the previous two chapters. A wide range of cells are capable of producing MMPs, including cardiac myocytes and infiltrating immune cells [5,6]. In particular, MMP-8 (collagenase-2) and MMP-9 (gelatinase B) modulate both the innate and the adaptive immune responses. The size and activity of MMP-8 are different from various sources, notably PMN-derived or mesenchymal cell-expressed. In PMNs, MMP-8 is synthesized as an 80 kDa latent enzyme and subsequently cleaved into a 75 kDa active protease, while smaller forms (45-55 kDa) have been observed in mesenchymal cells [7]. MMP-9 is one of the most extensively studied proteases and it exists as a 90-100 kDa latent enzyme which undergoes cleavage to an 82 kDa A version of this chapter has been submitted to Circulation for publication. Cheung C, Luo H, Yanagawa B, Leong H, Zhang J, Rahmani M, Walinski H, Walker E, McManus B. Ablation of MMP-9 in CVB3-infected mice increases severity of myocarditis. 149 active form. The expression of these two enzymes is induced usually by cytokines and growth factors ( T N F - a , TGF -P , and interleukins) but they are also produced and stored in granules within P M N cells. They can also be activated non-proteolytically by R O S and by other proteases, such as cathepsin G , chymotrypsin, and plasmin, as well as several M M P s ( M M P - 3 , M M P - 7 , M M P - 1 0 , and M M P - 1 4 ) [7,8]. Within the immune system setting, M M P - 8 and M M P - 9 balance the inflammatory reaction through both proteolytic activation, for example I L - l p [9], and inactivation, such as IFN-p [10], of cytokines and chemokines. M y previous results in association with other studies showed upregulation of M M P - 2 , -8, -9, and -12 during the inflammatory phase o f viral myocarditis, thus suggesting that these proteases may play a role in immune cell modulation [11-13]. In this study, I investigated the roles o f M M P - 8 and M M P - 9 during the pathogenesis of acute murine myocarditis following C V B 3 infection to better understand their immunomodulatory effects. 4.2 Materials and Methods 4.2.1 Experimental Groups The M M P 9 K O mice, a generous gift from Dr. Robert Senior (Washington University School o f Medicine. St. Louis, M O ) were produced by V u et al. and their W T counterpart is the 129SvEv strain (Taconic, Germantown, N Y ) [14]. The M M P 8 K O mice, a generous gift from Dr. Christopher Overall (Department of Oral Biological & Medical Sciences, U B C ) were generated as previously described and their W T counterparts (C57/B16) were homozygous +/+ derived from the same parental line as the null mutants from heterozygous pairings [15]. 150 4.2.2 Virus Infection and Detection Virus stocks were propagated by passaging in HeLa cells and viral titers were determined by standard plaque assay procedure as described in Chapter II [16]. Five week-old adolescent males from all four genotypes were infected intraperitoneally with 105 PFU of CVB3 (Gauntt strain) or PBS, and mice were sacrificed at 9 days pi. In total, 19 WT 129SvEv, 17 MMP9KO, 11 MMP8WT, and 11 M M P 8 K O mice were used in this study. A l l animals were weighed daily following infection, and their vital signs and morbidity states were recorded. Homogenized heart tissue was assessed using plaque assay for presence of virus, as previously described [17]. A l l animal procedures were in accordance with the Animal Care Committee, University of British Columbia. 4.2.3 Gelatin Zymography Heart tissue was assessed using gelatin zymography to detect the activity of MMP-2 and -9, as previously described in Chapter II [18]. 4.2.4 Western Blot Frozen apices were homogenized in NP-40 buffer with a glass homogenizer and lOOug of total protein was electrophoresed under reducing conditions through 10% polyacrylamide gels at 100V for 2 hours, as described in Chapter II. The proteins were transferred overnight onto nitrocellulose membranes and then the membranes were blocked with 5% skim milk powder in TBS-T. The following antibodies and dilutions were applied overnight at 4°C in 2.5% skim milk buffer: 1/3000 anti-MMP8 (gift from Dr. Overall), 1/1000 anti-MMP-12 (BIOMOL), and 1/500 anti-MMP13 (Neomarkers/Labvision, Fremont, CA). 151 4.2.5 Histological Assessment Processing and H & E staining o f cardiac tissue were as described in Chapter II. Images were all captured using a Nikon inverted microscope and Spot digital camera. 4.2.6 Immunohistochemistry Paraffin-embedded heart sections were immunolabeled for the presence and localization of the following proteins, using the indicated dilution of the antibodies: 1/100 C D 3 (Dako), 1/3000 neutrophil marker (clone 7/4, Serotec, Raleigh, N C ) , and 1/30 CD45 ( B D Biosciences, Mississauga, ON) . Briefly, slides were heated in 0 . 0 I M citrate buffer p H 6.0 using a microwave oven and incubated overnight at room temperature using the above-mentioned antibodies diluted in serum-free protein block (Dako), and detected using the . ABComplex amplification system with vector red (Vector Labs) as substrate. Images were all captured using a Nikon inverted microscope and Spot digital camera. 4.2.7 Quantitative Real Time Polymerase Chain Reaction (qPCR) Transcriptional expression of IFN-p\ IFN-y, I L - l p \ IL-4, IL-5, IL-6, IL-10, IL-12, MIP-loc, TGF-(3l , and T N F - a , following virus infection were assessed using qPCR. Total mouse R N A was isolated from the basal portion of the heart using RNeasy kit (Qiagen) and lp:g of R N A was converted to c D N A using Superscript reverse transcriptase (Invitrogen) according to manufacturer's protocol. Predesigned P C R primers and probes from the TaqMan® Gene Expression Assays collection were purchased from Applied Biosystems (Foster City, C A ) and the reaction conditions were run according to manufacturer's protocol on the Applied Biosystems 7900HT Fast R T - P C R System. 152 4.2.8 Data and Statistical Analyses Image-Pro Plus® program was used to quantitate the staining obtained by immunohistochemistry. Briefly, at least 10 images from each individual mouse was taken using a 2 0 X objective lens and the area of staining was measured using colour segmentation technique. The N I H ImageJ 1.3 l v program (http://rsb.info.nih.gov/ij/) was utilized to quantitate zymography band intensities. Internal control for the q P C R was performed with G A P D H . The data is presented as mean ± SD in the text and graphs. Pairwise comparisons between virus-infected W T and the corresponding K O mice were conducted using two-tailed, unpaired Student's t-test or the Wilcoxon non-parametric test and differences were considered significant at p<0.05. 4.3 Results 4.3.1 Gelatinolytic activation of MMP-2 and MMP-9 I confirmed the absence of M M P - 9 in the M M P 9 K 0 mice using gelatin zymography (Figure 4.1). I further observed that there was no compensation by M M P - 2 in the M M P 9 K 0 mice. The pro- and active forms of M M P - 2 were detected in all samples, whereas as expected, only the lOOkDa band of M M P - 9 was detected in W T , as previously described [11]. A s shown in Figure 4.1, the gelatinolytic activities of both proteases were upregulated in infected W T mice as compared to sham and as expected, only M M P - 2 was upregulated in the infected M M P 9 K 0 mice. However, even though the M M P 9 K 0 mice did not exhibit any M M P - 9 gelatinolytic activity, there appears to be no compensation by M M P - 2 as there was no difference between any o f the forms of M M P - 2 in W T and M M P 9 K 0 mice. 153 Figure 4.1: Zymography analysis of M M P - 2 and M M P - 9 activities in CVB3-infected M M P 9 K O and W T mice. (A) A representative blot is shown and an individual sample was run per lane. (B) Densitometric comparisons (mean±SD) of the M M P - 2 bands between M M P 9 K O and W T using the ImageJ program. Results are expressed as intensity in arbitrary units. A Sham Infected WT MMP9KO WT MMP9KO MMP9 proMMP2 active MMP2 B 1400 1200 >/| IOOO I £ 800] 1 g 600 J 400 w 200 0 MMP-9 SHKO SH WT 9V KO 9V WT MMP-2 • Pro • Active SH KO SH WT 9V KO 9V WT 154 4.3.2 Protein Expression of MMPs To further determine whether there is compensation by other M M P s , I next investigated the protein expression of M M P - 8 , M M P - 1 2 , and M M P - 1 3 by Western blotting. Figure 4.2 shows that the mesenchymal-derived forms of M M P - 8 (45-55kDa) were predominantly detected and active M M P - 8 (45kDa) was strongly expressed in both W T and M M P 9 K O mice, the quantitation of which is graphed in Figures 4.2A and 4.2C showing no significant difference between the two groups [19]. Pro (55 kDa) and active M M P - 1 2 (45 kDa) as well as a higher molecular weight band were expressed in comparable amounts in both W T and M M P 9 K O mice, as shown by the densitometric graph in Figures 4.2A and 4.2D. Three forms of M M P - 1 3 can be detected, the glycosylated proenzyme (70 kDa), unglycosylated proform (55 kDa), and the active M M P - 1 3 enyzme (40 kDa), and all three bands did not differ between W T and M M P 9 K O , as shown in Figures 4.2A and 4.2E [20]. Next I investigated the expression of all four TIMPs, the specific endogenous inhibitors of M M P s . A s shown in Figure 4.2B, all four TIMPs were detected in both M M P 9 K O and W T mouse hearts but T IMP-2 ( 0 . 8 ± 0 . 2 in M M P 9 K O vs. 1.1±0.3 in W T , p=0.04) and TIMP-3 (1.0±0.3 in M M P 9 K O vs. 1.3±0.4 in WT,/?=0.04) were significantly decreased in M M P 9 K O as compared to W T and there was a trend towards TIMP-1 downregulation as well (2.2±0.6 in M M P 9 K O vs. 3.0±1.3 in W T , p=0.09). The quantitations of these proteins are graphed in Figure 4.2F. Therefore, even though the expression and activation o f M M P s were comparable between W T and M M P 9 K O mice, downregulation of T IMPs may result in an imbalance o f protease inhibition, thus resulting in increased M M P activity. 155 Figure 4 . 2 : Translational expression of MMPs and TIMPs in M M P 9 K O and W T mouse hearts. Ixnmunoblotting for (A) M M P - 8 , M M P - 1 2 , and M M P - 1 3 and (B) all 4 TIMPs revealed no significant differences in the expression of these enzymes between M M P 9 K O and W T mice. A representative blot is shown for each protein and an individual sample was run per lane. Densitometric comparisons (mean±SD) of (C) M M P - 8 , (D) M M P - 1 2 , (E) M M P - 1 3 , and (F) T IMPs expression levels between M M P 9 K O and W T using the ImageJ program showed no significant difference between any of the forms. Results are expressed as intensity in arbitrary units. N=8-12 mice per assay. ¥ p<0.05 A B 190-120-85-60-50-40-25-K W K W K W K W mm mm . v.v.-. ^ , MMP8 MMP 12 MMP 13 GAPDH K W — — TIMP-1 TIMP2 — TIMP-3 TIMP-4 mm — GAPDH c D MMP8 MMP 12 • TOTAL E l 55kDa • 45kDa 35kDa B 30kDa • 22kDa • TOTAL | 85kDa • 55kDa • 45kDa MMP9KO WT MMP9KO WT E 4 3 2 .1? 1 'so h & 0 MMP 13 • TOTAL • 70kDa = 55kDa • 35kDa : D 3  lluniv. a D i H 1 Si a TIMPs • TIMP-1 • TIMP-2 B TIMP-3 • TIMP-4 MMP9KO WT MMP9K0 WT 156 Figure 4.3: M o r b i d i t y assessment of CVB3-infected MMP9KO, MMP8KO, and control mice at 9 days p i . Histological H & E stains of (A) sham M M P 9 K O and M M P 9 W T mice, (B) CVB3-infected M M P 9 K O and M M P 9 W T mice, (C) sham M M P 8 K O and M M P 8 W T mice, and (D) CVB3-infected M M P 8 K O and M M P 8 W T mice. (E) Quantitation of the histological scores comparing M M P 9 K O and W T mice. (F) Vi ra l load within M M P 9 K O mouse hearts was higher than WTs , as determined by plaque assay. Log scale is shown in graph. (G) Quantitation of the histological scores comparing M M P 8 K O and W T mice. (H) Plaque assay results of M M P 8 K O and W T mouse. Data shown as mean ± SD. A l l animals were used for assessment. #/?<0.005, ¥/?<0.05. Sham B Infected M M P 9 K O WT M M P 9 K O WT Sham Infected 157 4.3.3 Morbidity and Histological Examination There were no significant cardiac alterations between the M M P deficient mice and their corresponding W T , as shown in Figures 4.3A and 4.3C. The M M P 9 K O mice appeared to suffer more severe disease as reflected by their morbidity states and histological scoring o f the H & E stains for cardiac injury (Figures 4.3B and 4.3E) despite similar mortality rates (13% for M M P - 9 K O and 10% for W T mice, data not shown). Pathological scoring o f H&E-stained myocardium showed that the M M P 9 K O suffered more severe cardiac damage and inflammation as compared to W T (3.6±0.9 vs. 2.4±1.3, ^=0.005), as summarized in Figures 4.3E. In the M M P 9 K O mouse hearts, there appeared to be more myocytolysis, calcification, and cellular infiltrates. Plaque assay was performed to determine the presence and amount o f virus within CVB3-infected hearts (Figure 4.3F). M M P 9 K O had more viral load, approximately half a unit on a loglO scale, p=0.04, within the myocardium following C V B 3 infection than the W T . Despite the fact that M M P 8 K O appeared to have increased morbidity, as measured by body weight, which showed that M M P 8 K O mice experienced a higher percentage of body weight loss (data not shown), there was no significant difference in mortality and degree o f cardiac injury between the M M P 8 K O and W T mice (Figure 4.3D and 4.3G). This corresponded to the amount of virus within the hearts, as measured by plaque assay, which also showed no significant difference between M M P 8 K O and W T mice (Figure 4.3H). 158 Figure 4.4: Analysis of fibrosis in CVB3-infected MMP9KO, MMP8KO, and control mice at 9 days pi. Shown here are representative light microscopic images o f (A) picrosirius red-stained hearts from M M P 9 K O and M M P 9 W T mice and (B) quantitation of collagen volume fraction in these mice. (C) Picrosirius red staining and (D) quantitation of collagen volume fraction were performed in M M P 8 K O and M M P 8 W T mice. Ye l low denotes muscle fiber and red is collagen. N=8-12 animals per genotype. ¥p<0.05. Sham M M P 9 K O W T Infected M M P 9 K O W T B D M M P 9 K O W T M M P 8 K O W T Sham Infected if i M M P 8 K O W T M M P 8 K O W T 159 Picrosirius red stain was used to detect fibrosis. Sham hearts were virtually identical between M M P 9 K O and M M P 9 W T mice (Figure 4.4A). Virus-infected M M P 9 K O hearts stained with picrosirius red revealed a marked increase in collagen deposition as compared to W T s (10.6±2.7% vs. 7.1±2.6% respectively, p=0.04). Due to large areas o f myocyte dropout in the myocardium o f M M P 9 K O mice, there was abundant replacement fibrosis while reactive fibrosis also occurred in interstitium adjacent to viable cells. A common observation in both groups of animals is perivascular fibrosis (arrow). Morphometric analyses of these results are summarized in Figure 4.4B. Sham hearts were virtually identical between M M P 8 K O and W T mice as well (Figure 4.4C). After virus infection, collagen accumulation could be detected in areas o f myocyte dropout. Perivascular and reactive fibrosis were also detected albeit these were not common events in both genotypes. There was no significant difference in the amount nor in the quality of the collagen between the M M P 8 K O and W T hearts (Figure 4.4D). 4.3.4 Immune Infiltration in KO and WT Mouse Hearts A major role of M M P - 9 and M M P - 8 is immune regulation, by modulating the migration and activity of various immune cells and cytokines. Therefore, I compared the amount of infiltration of total leukocytes (CD45 a pan-leukocyte marker), phagocytes ( P M N s and macrophages), and T-lymphocytes (CD3, a pan-T-cell marker), which are the major immune cell subtypes in myocarditis, in W T and K O mice. The amount of total immune infiltration was increased in M M P 9 K O mice as compared to W T (15.2±12.6% vs. 2.0±3.0% respectively, /?=0.002), as measured by CD45 immunostaining. Immunopositivity in the M M P 9 K O consisted mostly o f focal lymphoid 160 aggregrates localized to areas of extensive injury while in the W T mice, the inflammatory foci were smaller and more infrequent as shown in Figures 4.5A and 4.5B. Next I performed immunostaining for phagocytes, which are innate cells responsible for the first line of defense against viruses and initiation of wound repair. Both neutrophils and macrophages were detected in the myocarditic myocardium and we observed no apparent difference in infiltration of these subtypes between M M P 9 K O and W T mice (2 .7±2.5% vs. 1.5±1.5% respectively, p-0.26). Figures 4.5C and 4.5D show representative micrographs from each genotype. Lastly, I investigated the infiltration of T-cells in myocarditic M M P 9 K O and W T hearts. Immunostaining for C D 3 was significantly increased in M M P 9 K O animals as compared to controls, 4 .2±2.0% vs. 1.8±1.5% respectively, /?=0.005. C D 3 is found on all T-cells regardless o f activation status. In both genotypes, majority of the T-cells were localized to inflammatory foci with a few diffusely stained cells. Figures 4.5E and 4.5F show representative micrographs from each genotype. Morphometric analyses o f the immunostaining for CD45, phagocytes, and CD3 in M M P 9 K O and W T are represented in Figure 4.5G. I also performed immunostaining for phagocytes and C D 3 in M M P 8 K O and their corresponding control mice. In these mice, phagocytic infiltration into the myocardium was not significantly different (2 .2±0.5% vs. 3.2±0.5% respectively, p=0.6) and the phagocytes were localized to similar sized lesions in the heart (Figures 4.5H and 4.51). However, T-cell infiltration into the myocarditic myocardium of M M P 8 K O mice was significantly decreased as compared to W T mice (0.9±1.0% vs. 7.2±6.9% respectively, /?=0.0006) as shown in Figure 4.5J and 4.5K. The T-lymphocytes localized mostly to inflammatory lesions in the W T mice 161 while in the M M P 8 K O , the T-cells were more diffusely situated. Morphometric analyses of the immunostaining for phagocytes and C D 3 in M M P 8 K O and W T are represented in Figure 4.5L. 162 Figure 4 . 5 : Immunohistochemistry staining for C D 4 5 , phagocytes, and T-cells in C V B 3 -infected M M P 9 K O , M M P 8 K O , and control mice. Immunostaining for CD45 in (A) M M P 9 K O and (B) W T mice; phagocytes in (C) M M P 9 K O , (D) M M P 9 W T , (H) M M P 8 K O and (I) M M P 8 W T ; and T-cells in (E) M M P 9 K O , (F) M M P 9 W T mice, (J) M M P 8 K O and (K) M M P 8 W T mice. Quantitation of immunostaining in (G) M M P 9 K O and (L) M M P 8 K O mice and their corresponding controls by Image Pro Plus are represented. N=8-12 animals per genotype. # /?<0.005, % /K0 .02 . 163 4.3.5 Transcriptional Regulation of Cytokines I then looked at the transcriptional regulation of cytokines to see i f the differences in immune cell distribution were due to an imbalance in cytokine expression. I used q P C R to compare the transcription of IFN-p, IFN-y, I L - l p , IL-4, IL-5, IL-6, IL-10, IL-12, M l P - l a , T G F - p i , and T N F - a , which previous studies have shown to play important roles in myocarditis and wound healing, between W T and M M P 9 K O animals. In the infected M M P 9 K O mouse hearts, IFN-p 1 (KO=1.36±1.35 vs. WT=0.32±0.69, p=0.003), INF-y(KO=0.22±0.19 vs. WT=0.10±0.07, p=0.04), IL-6 (KO=0.50±0.72 vs. WT=0.09±0.07, p=0.01), IL-10 ( K O 0 . 2 5 ± 0 . 1 4 vs. WT=0.12±0.08,/?=0.04), and M l P - l a ( K O = 1 . 5 5 ± 1 . 2 8 vs. WT=0.65±0.56, p=0.04) were all increased as compared to controls. There was an upward trend for IL-l(3 expression albeit it did not reach statistical significance (KO=0.95±0.08 vs. WT=0.90±0.03 , p=0.06). T N F - a , which has been shown to play a major role in many heart failure models and is a potent regulator o f wound healing, was not significantly different between M M P 9 K O and W T mice. Figure 4.6 shows the analyses of these cytokine transcripts, measured as arbitrary units and normalized to G A P D H expression. 164 Figure 4.6: Transcriptional analysis of cytokines in MMP9KO and WT mice. Quantitative P C R was performed to detect the cytokine profiles in CVB3-infected M M P 9 K O , M M P 8 K O , and control mice at 9 days pi . The results are shown as relative abundance as compared to G A P D H . We detected upregulafion of IFN-P, IFN-y, I L - l p , IL-6, IL-10, and M l P - l a . # ;?<0.005, % p<0.02, ¥ /?<0.05. • IFN-p 1 • IFN-y • I L - l p K O W T 4.4 Conclusions and Discussion In this study, we infected two different strains of M M P deficient mice, M M P 9 K O and M M P 8 K O , and compared cardiac injury, viral load, and inflammation between the K O s and their corresponding WTs. M M P 9 K O mice experienced more severe cardiac injury, virus abundance, fibrosis, and inflammation as compared to controls. Upon dissection of the immune infiltrates, we observed an increased migration of T-cells into the myocarditic M M P 9 K O mice with no difference in phagocytic infiltration. This corresponded to increases in various cytokines including IFN-p, IFN-y, I L - l p , IL-6, IL-10, and M l P - l a . In contrast, 165 M M P 8 K 0 mice suffered the same degree of cardiac injury, fibrosis, and viral infection as their W T counterparts. However, upon inspection of the immune cell subtypes, we observed a decrease in T-cell infiltration into the M M P 8 K O myocardium as compared to controls while the amount o f phagocytes was equivalent in both genotypes. These results contrast other studies, in which M M P - 9 was shown to be associated with maladaptive remodeling in both animal models and humans studies of M I , hypertension-induced L V hypertrophy, myocardial stunning, myocarditis, and heart failure, while M M P - 9 deficiency contributes to an improvement in cardiac remodeling and maintenance o f heart structure and function [21-23]. In patients presenting with L V hypertrophy, M M P - 9 was increased in plasma and this correlated to L V diastolic wall thickness and Doppler indices o f diastolic dysfunction [24]. In addition, M M P - 9 levels were also associated with increased L V diastolic dimensions, increased wall thickness, lower L V E F , and higher end-systolic volumes (ESV) in patients following an M I attack and with H F [23,25]. Less is known about M M P - 8 and its effect on cardiac diseases. M M P - 8 has been shown to be upregulated in animal M I models, human patients following an ischemic attack, and atherosclerotic disorders but the definite role o f this enzyme in myocardial disorders is not clear [7,22,26,27]. In all o f these diseases, the primary role of M M P - 8 and M M P - 9 is suggested to be in modification o f the matrix. During M I , breakdown of the collagen network begins within minutes of the ischemic injury, possibly through induction of M M P s by R O S and cytokines, which continues for days afterward. Infiltration o f innate immune cells, such as P M N s , contributes to further synthesis and activation of these M M P s , which continue to degrade the matrix. The matrix disruption is subsequently followed by an increase in collagen accumulation, which initially sustains the structure and function of the heart but progressive 166 fibrosis reduces proper contractility while continuous remodeling can lead to excessive matrix degradation by M M P s , thinning of the ventricular walls, cardiac dilatation, and ultimately heart failure [28,29]. Investigators also found similar mechanisms in hypertrophy, myocardial stunning, and induced-tachycardia models. Experimental deletion of M M P - 9 in mice reduced the relative incidence of myocardial rupture and attenuated L V dilatation in the first 2 weeks after M I [30,31]. Loss of M M P - 9 in hypertensive mice also modestly improved cardiac function, decreased fibrosis, and cardiomyocyte hypertrophy up to 7 weeks following the onset o f injury [32]. M M P - 8 has not been individually investigated in myocardial disorders but this enzyme has been shown to be increased within myocardial infarct tissue and abdominal aortic aneurysms that may predispose them to rupture due to excessive collagen degradation [33-35]. Despite the plethora of studies of M M P s in M I , hypertrophy, and H F , the roles o f M M P s in inflammatory cardiac diseases are not well elucidated. Previous studies performed by our laboratory and others have shown that there was an imbalance in the expression and activation o f M M P s and TIMPs following C V B 3 infection, whereby M M P s - 2 , -9, and -12 were upregulated and TIMPs were downregulated during the early (day 3), inflammatory (day 9), and late (day 30) phases of viral myocarditis in mice [11,12,36]. In addition, treatment o f CVB3-infected mice with a beta-adrenergic receptor blocker, carvedilol, decreased inflammation and fibrosis, possibly through downregulation o f M M P - 8 [37]. Evidence from studies o f M M P - 8 and M M P - 9 in other cardiac disease models prompted investigators to suggest that the role of these M M P s was predominately in E C M degradation during viral myocarditis. However, these two enzymes have been shown to play important roles in the immune system, notably innate immunity; both proteases are found within granules in 167 neutrophils and have been suggested to be major effectors of this cell type. In addition, M M P - 9 and M M P - 8 are known to be expressed by all migrating cell types, including stem cells, lymphocytes, and fibroblasts. M y observations using CVB3-infected M M P - 9 and M M P - 8 deficient mice showed contrasting results to other cardiac disease models, in that the loss o f M M P - 9 resulted in increased disease progression and in both K O mice, there was differential T-cell infiltration. This difference could be explained by the fact that myocarditis is an inflammatory disease caused by infection while the other models have different etiologies. In bacteria-induced peritonitis and meningitis models, the loss of M M P - 9 increased the severity o f disease and bacterial titers through impairment of the immune response [38,39]. In Staphylococcus aureus-thggered septic arthritis in M M P 9 K O mice, severity of the disease, bacterial titers, and immune cell infiltrates into the affected organs were increased, possibly due to impairment of the immune response during the early phase of the infection, which resulted in delays in microbial clearance and reparative mechanisms, leading to an increased late immune response [40]. This study indicated that in C V B 3 -infected M M P - 9 deficient mice, there was increased viral titer despite an increase in immune cell infiltration, specifically T-lymphocytes, which suggests that impairment of the innate immunity lead to incomplete elimination of the virus and delayed inflammation. The frequency of phagocytes, which belong to the innate immune response, was not significantly different between M M P 9 K O and W T mice, but the activity and function of these cells may have been affected. Further, the amount of P M N s and macrophages was not individually measured in this study. Therefore, more detailed investigations of the activity, function, and immune subtypes during an earlier timepoint (1-3 days pi) would clarify the importance of the innate immune response in myocarditis. In contrast, I did not observe any significant 168 difference between the severity and overall inflammation between M M P - 8 K O and W T mice, although a significant decrease in T-cells was detected in the M M P 8 K O mice. This suggests that in viral myocarditis, the role of M M P - 8 lies not in innate immunity but in modulation of the adaptive immune response. Both M M P - 9 and M M P - 8 have been shown to be involved in cytokine and chemokine processing, which would directly affect the recruitment, activation, and function of immune cells. Previous reports showed that these two enzymes have similar C X C chemokines substrates, including monokine induced by IFN-y (MIG), IFN-inducible protein-10, and L P S -induced C X C chemokine (LIX) , whereas M M P - 9 can further digest platelet factor-4, G R O - a , and connective tissue-activating peptide-III [41]. In addition, M M P - 9 can regulate the synthesis and clearance of I L - l (3 by cleavage of the inhibitory domain of I L - l (3 to generate biologically active cytokine as well as proteolytic inactivation of this molecule, a process that is modulated by the balance of M M P s , TIMPs, and cytokines [42,43]. This enzyme can cleave the latent TGF-binding protein (LTBP) and proIL-8 to generate catalytically active cytokines, which in turn can further upregulate the transcriptional expression of M M P - 9 [44-47]. In addition, pro tease-mediated inactivation of interleukin receptors (IL-2 and IL-5) and IFN-P may limit the activities of these cytokines, resulting in downregulation of immune cell activities [48,49]. I observed increased transcriptional expression o f IFN-P, IFN-y, IL-6, IL-10, and M l P - l a while I L - l p , IL-4, IL-5, I L - l 2 , T N F - a , and T G F - p l remained unchanged in CVB3-infected M M P 9 K O mice as compared to W T mice. This is consistent with ongoing inflammation and persistent viral infection, since the viral titer is still high. However, further investigations into the post-translational modifications of the cytokines using protein 169 detection and activation assay methods are required to determine the effect of M M P - 9 on these cytokines. M y results suggest that M M P - 9 is uniquely involved in a complex network for cytokine/chemokine and ultimately immune cell regulation, whereby this enzyme is necessary for proper inflammatory control because infected M M P 9 K O did not have compensatory increases in M M P - 2 , M M P - 8 , M M P - 1 2 , and M M P - 1 3 , which suggests that the investigated enzymes exhibited no redundancy for M M P - 9 . However, downregulation o f T I M P - 1 , T I M P -2, and TIMP-3 in M M P 9 K O mice suggests that the overall M M P activity level is perturbed, resulting in an increase in collagen deposition, as viewed using picrosirius red staining. The downregulation of T IMPs may be due to a negative feedback loop, in which a deficiency in M M P - 9 may have decreased the pool of total M M P s in our model, in turn decreasing the pool of TIMPs . Many studies have shown the importance of TIMP-1 and TIMP-3 in cardiac remodeling: overexpression o f TIMP-1 in mice with hypertension reduced cardiac hypertrophy, fibrosis, L V dilatation, and preserved L V function while TIMP-3 K O mice experienced spontaneous L V dilatation, cardiomyocyte hypertrophy, and L V dysfunction in correlation to an increase in M M P - 9 levels with aging and in pressure overload hypertrophy [32,50,51]. Further, Heymans et al. overexpressed TIMP-1 in CVB3-infected mice and observed considerable amelioration of inflammation and cardiac injury [21]. These reports correspond to my results shown in this chapter, in which downregulation of TIMPs is associated with increased severity of myocarditis. The decrease in TIMP-2 may trigger an increase in M M P activities because the activation and inhibition of M M P - 2 is dependent on TIMP-2 , whereby bantam amounts of TIMP-2 induces activation of M M P - 2 in conjunction with M T 1 - M M P but increased expression of TIMP-2 inhibits both M M P - 2 and M T 1 - M M P 170 [52,53]. A n increase in overall M M P activity may trigger collagen accumulation, although this appears counterintuitive, but M M P s are able to regulate various pro-fibrotic pathways due to their broad substrate specificity. Several M M P s , including M M P - 3 , and M M P - 9 are capable of cleaving the L T B P to free active T G F - p l , the predominate pro-fibrotic cytokine which directly stimulates myofibroblasts to increase collagen deposition and suppresses M M P expression [54-56]. We did not observe any difference in T G F - p i transcriptional expression in M M P 9 K O as compared to controls but it is necessary to assay for active T G F - p i using E L I S A or Western blot techniques to clarify the activity level of T G F - p i in M M P 9 K O mice. In addition, active endothelin-1 (ET-1), a potent vasoconstrictor and pro-fibrotic molecule that directly stimulates myofibroblast proliferation and collagen deposition, can be generated by M M P - 2 dependent cleavage of its precursor big ET-1 [57]. ET-1 can also regulate the expression o f M M P s by direct repression (MMP-1) or stimulation ( M M P - 2 and M M P - 9 ) o f their expression [58]. In contrast, no differences in collagen deposition and organization were-observed between the M M P 8 K O and W T mice, further suggesting that this enzyme functions during late remodeling and immunomodulation. In this study, I compared two M M P s that are both involved in collagen regulation and immunomodulation. 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Heart Fai l Rev 2004;9:53-61. 176 CHAPTER V: NEUTRALIZING ANTI-4-1BBL TREATMENT IMPROVES CARDIAC FUNCTION IN VIRAL MYOCARDITIS 5.1 Rationale The last chapter showed evidence that differential T-cell regulation can lead to drastic results, as observed by increased T-cell infiltration and severity of myocarditis in M M P 9 K O mice. Interestingly, decreased T-cell infiltration was detected in M M P 8 K O mice yet the severity of disease was comparable to control animals, suggesting that effects o f this immune dysregulation cannot be observed at the timepoint we investigated. Therefore, in order to better understand the role of T-lymphocyte infiltration, I decided to examine the inhibition o f these lymphocytes during the chronic phase of myocarditis. The 4 - I B B pathway belongs to the tumour necrosis factor receptor (TNFR) family and consists of the receptor 4 - I B B and its ligand 4 - 1 B B L . It has been studied extensively in the immune system where they were first discovered as co-stimulatory molecules involved in T-cell activation [1]. The 4 - I B B antigen is a cell membrane protein, which is usually induced on activated T-cells and acts to influence effector functions and T-cell numbers in the late stage o f the acquired immune response [2]. The most characterized ligand for this protein is 4 - 1 B B L , which is constitutively expressed on plasma monocytes and dendritic cells but can also be induced on B-cells, activated macrophages, as well as cardiomyocytes [3,4]. Stimulation of 4 - 1 B B L expression can occur by C D 4 0 / C D 4 0 L ligation, L P S , and other toxic and infectious insults [5]. Upon activation, 4-lBB-expressing cells upregulate the A version of this chapter has been accepted for publication in the July edition of Laboratory Investigation. Cheung C, Deisher T, Yanagawa B, Luo H, Bonigut S, Samra A, Zhao H, Walker E, McManus B. Neutralizing anti-4-lBBL treatment improves cardiac function in viral myocarditis. 177 secretion of cytokines, such as IL-6, IL-8, and T N F - a [5,6]. The 4 - I B B signaling pathway plays crucial roles in the enhancement of integrin-mediated cell adhesion, regulation o f T-cell effector functions, and prevention and/or stimulation o f cell death of leukocytes [7-9]. In contrast to it's role as a survival factor for activated CD8+ T cells, 4 - I B B has also been implicated in activation-induced cell death in CD4+ T cells, as well as stimulation of apoptosis in neutrophils and B-cells [4,10,11]. Previously, initiation of cell death through the CD95 (Fas, APO-1) pathway and subsequent activation of effector caspases had been suggested but recent studies have shown involvement of alternate mechanisms [4,12]. Recently, the importance of these co-stimulatory molecules has been shown in the pathogenesis of myocarditis and heart failure. Both 4 - I B B receptor and its ligand are upregulated in cardiomyocytes in C V B 3 infection [13], chemical-induced cardiotoxicity, and ischemia (unpublished data), in both a murine model and in patients [14,15]. A study by Seko et al. reported that inhibition of the 4 - I B B pathway significantly attenuated inflammation and myocardial injury during the acute phase o f myocarditis in mice but the functional consequences are yet unknown [13]. Our research has demonstrated that blockade of the 4 - I B B pathway improves cardiac function in doxorubicin-induced cytotoxic models and acute M I . Furthermore, agonism of the 4 - I B B receptor exacerbates cytotoxic cardiac damage. In vitro studies demonstrated that 4 - I B B can be expressed on injured cardiomyocytes and induces apoptosis via mitochondrial disruption. In this study, I investigated the role of T-lymphocytes in chronic myocarditis and demonstrated the effect of 4 - I B B pathway inhibition on cardiac function by treating C V B 3 -infected mice with a neutralizing monoclonal an t i -4 - lBBL antibody. 178 5.2 Materials and Methods 5.2.1 Virus Infection and Animal Treatment Vira l stocks were propagated by passaging through HeLa cells and titers were routinely determined by standard plaque assay procedure as described in Chapter II [16]. A pilot study was performed to determine the course of cardiac changes after C V B 3 infection. For this study, five week-old male A / J mice were infected with 10 5 P F U of cardiovirulent C V B 3 (Gauntt strain) or sham-infected with P B S and four mice per day were sacrificed on days 3, 9 and 30 pi . In the chronic study, five week-old adolescent male A / J mice (Jackson Laboratories) were infected with C V B 3 or sham-infected with P B S and at 14 days p i , mice were assigned to the following treatment groups: C V B 3 and neutralizing a n t i - 4 - l B B L A b [M522] (n=9); C V B 3 and non-blocking an t i -4 - lBBL antibody [M525, control] (n=10); sham and neutralizing M522 (n=12); and sham and non-blocking control antibody (n=12). Treatments were started on day 15 pi during the inflammatory resolution by weekly intraperitoneal injections of 250 jig neutralizing M522 or non-blocking control A b . Antibodies were produced in rats and affinity-purified for rat IgG2a isotypes (Amgen, Inc.). A l l surviving mice were sacrificed at 10 weeks pi . This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the U S National Institutes of Health (NIH Publication No . 85-23, revised 1996) and the Animal Care Committee o f University o f British Columbia. 5.2.2 Fluorescent Immunohistochemistry Cardiac tissue was fixed in 10% formalin and embedded in paraffin. Serial sections of mid-ventricular heart tissue were deparaffinized in subsequent washes of xylene and alcohol and blocked with universal protein block (Dako). The slides were treated with 0 .2M 179 glycine for 20 minutes. For fluorescent detection of 4 - I B B , I used l | i g / m L o f 4 - 1 B B L antigen fused to a leucine zipper (LZ) motif (Amgen, Thousand Oaks, C A ) followed by Alexa Fluor® 488 labeled anti-LZ antibody. For detection of 4 - 1 B B L , l|a,g/mL o f human Fc-linked 4 - I B B antigen was applied followed by Alexa Fluor® 594-labeled anti-human immunoglobulin (Molecular Probes/Invitrogen) secondary antibody. Stained sections were viewed using the Leica S P 2 A O B S confocal laser scanner on a Leica D M IRE inverted microscope. A t least 10 images were taken from each sample at 2 0 X magnification and morphometric analyses, using Image-Pro Plus, were performed to calculate the percentage of staining. 5.2.3 Echocardiography M i c e were anesthetized with ketamine-xylazine (100 mg/kg-7.5 mg/kg xylazine) and echocardiograms were recorded using the S O N O S 5500 (Philips Medical , Bothell, W A . ) ultrasound machine and a pediatric S-12 phased array probe at the maximum frequency. For the pilot study, echocardiography was performed on mice at days 0, 3, 9 and 30 pi . For A b treatment groups, mice underwent echocardiography at day 9 and then weekly sessions from 4 to 10 weeks pi . Echocardiographic measurements were taken from 2D parasternal long axis ( P S L A ) and 2D short axis views at the mitral valve ( M V ) and papillary muscle (PM) levels. The mice laid prone on a 1 cm thick agarose gel pad on a temperature-controlled modified microscope stage. Views were standardized serially and between mice by strict adherence to the anatomical guidelines and conventions established by the American Society of Echocardiography. Measurements were taken at both diastole and systole and included: L V internal dimensions, L V area at the level of the mitral valve, L V area at the level of the 180 papillary muscles, and posterior wall (PW) thickness. Cardiac output (CO), ventricular volumes, and E F were calculated utilizing the Modified Simpson's algorithm. 5.2.4 Histological Examination Paraffin-embedded mid-ventricular tissue was sectioned and processed for H & E staining, to detect myocardial injury and inflammatory infiltration, and picrosirius red staining for collagen amount and orientation. Stained sections were viewed using light microscopy and graded in a blinded manner. A t least 10 images were taken from each sample at 2 0 X magnification and morphometric analyses, using Image-Pro Plus, were performed to calculate the percentage of picrosirius red staining. 5.2.5 RT-PCR R T - P C R was used to detect the transcript levels of M M P - 2 , -9, and -12 after virus infection and 4 - 1 B B L antibody treatment. Total mouse R N A was isolated from the basal portion of the heart using the RNeasy kit (Qiagen) and 0.5|J.g of R N A was converted to c D N A using Superscript reverse transcriptase (Invitrogen) according to manufacturer's protocol. The P C R primers and conditions are described in Chapter II [17]. For normalization purposes, 18S was used as control. 5.2.6 Immunohistochemistry For detection of CD45 and C D 3 , epitopes were uncovered by heating slides in 0.1 M citrate buffer p H 6.0 followed by overnight incubation in 20 \xg/mL o f anti-CD45 antibody ( B D Biosciences) and anti-CD3 antibody (Dako), as described in Chapter IV . The ABComplex amplification system (Dako) in conjunction with D A B was used for detection o f immunopositivity. Images were captured using a Nikon E600 inverted microscope and Spot digital camera, and morphometric analyses were performed using the Image-Pro Plus program. A t least 10 images were taken from each sample at 2 0 X magnification. 5.2.7 Data and Statistical Analyses A l l data were expressed as mean ± S.D. Pairwise comparisons between M522 and control samples were conducted using two-tailed, unpaired Student's t-test and Mann-Whitney U-test, and multiple comparisons were analyzed with A N O V A and Tukey's post-hoc test, with p<0.05 indicative of statistical significance. 182 Figure 5.1: Co-localization immunohistochemistry staining for 4-IBB and 4-1BBL in sham and CVB3-infected mice. Representative myocardial sections from (A) sham, (B) infected and untreated mouse at day 9, (C) M522-treated, and (D) M525 control animals showing staining for 4 - I B B (green, top small panels) and 4 - 1 B B L (red, lower small panels). The large panels represents overlay o f nuclear staining (blue) and the two stains (orange) is co-localization of the two antigens and blue represents nuclei. (E) Negative controls for 4-1BB and 4 - 1 B B L using isotype matched IgG. (F) Morphometric quantitation of 4 - I B B and 4 - 1 B B L protein expression. Scale bars=50^m. Data are mean ± SD. A B M522 CONTROL 183 5.3 Results 5.3.1 Immunohistochemical Staining for 4-IBB and 4-1BBL Previous observations have shown that 4 - I B B and 4 - 1 B B L can be induced in cardiomyocytes after various stimuli, such as doxorubicin treatment and myocardial infarction (unpublished data). Seko et al. also reported an induction of 4 - 1 B B L expression in cardiomyocytes following C V B 3 infection [13]. Therefore, I first examined the presence and amount of these two proteins in sham and CVB3-infected hearts at 9 days pi . Figure 5.1 A demonstrate minimal expression of the 4 - I B B (green, top small panel) receptor in sham mouse hearts and strong expression of 4 - 1 B B L (red stain, lower small panel) whereas at 9 days pi (Figure 5.IB), there was increased expression of 4-1BB (p<0.01) in infected hearts as compared to shams, which co-localized with cardiomyocytes (long arrow). The receptor and ligand co-localized predominantly in cardiomyocytes but were also expressed by small round cells, possibly immune cells (short arrow). In some areas of the myocardium, both 4 - I B B and 4 - 1 B B L were co-expressed in cardiomyocytes (orange colour, large panel). To confirm target coverage of the an t i -4 - lBBL antibodies (M522 and control Ab) into the mouse hearts, immunostaining for rat IgG2a was performed and immunopositivity was observed in all samples (data not shown). Immunofluorescent staining for 4 - I B B and 4-1 B B L at 10 weeks pi (Figures 5.1C and 5.ID) showed myocardial co-localization. A t 10 weeks p i , 4 - 1 B B L (red stain) was still strongly expressed in all tissue and no difference was found between the M522-treated (Figure 5.1C) and control hearts (Figure 5.ID, p=0J). In contrast, there was diffuse expression of 4 - I B B and for the most part it was co-expressed with its ligand. Both cardiomyocytes and small globular cells, possibly infiltrating cells (short arrows), expressed 4 - I B B whereas mostly endogenous cells expressed 4 - 1 B B L . In 184 M522-treated hearts, a trend towards increased 4 - I B B immunopositivity was detected as compared to the control group although the difference did not reach statistical significance (p=0;14). Figure 5.IF shows the graphical representation of the densitometric analyses. 5.3.2 Echocardiography Serial cardiac function measurements were taken by echocardiography. Figure 5.2A demonstrates the histological images at the various timepoints (top four panels) and the mid-section panels show the tracings for systolic short axis ventricular measurements while the bottom panels demonstrate diastolic short axis ventricular measurements. A t day 3 post-C V B 3 infection, histological myocyte vacuolization was associated with a non-significant trend in reduced L V wall thickening but normal wall thickness, E F , and cardiac volumes in virus-infected mouse hearts as compared to sham and baselines, which were measurements taken from the infected mice before infection (Figures 5.2B to 5.2E). B y day 9 p i , we observed a decline in the degree of wall thickening from 75% in sham hearts to 30% in infected myocardium (p<0.01) and 25-50% increase in wall thicknesses (p<0.01), suggestive o f wall edema and inflammation. Due to the increased wall thicknesses in the infected hearts, cardiac volumes were reduced by 40% at day 9 pi (p<0.01). On day 30 pi , wal l contractility remained depressed at 30% (p<0.01), diastolic wall thickness was still increased by 33% (p<0.01), the ejection fraction was reduced to 60% (pO.Ol ) , and systolic volume was increased by 33% (p<0.01), indicative of intrinsic contractile deficits. To examine chronic cardiac dysfunction and to determine the role o f the 4 - I B B pathway, I conducted a therapeutic treatment study and extended my observations to 10 weeks pi . Based on the baseline functional changes following C V B 3 infection, ultrasound was performed on day 9 pi and mice were normalized into two groups. On day 15 pi , one 185 group was administered a 4 - 1 B B L neutralizing antibody (M522), and the other group was given a control non-blocking antibody, M525 (control). Sham mice with neutralizing and non-blocking antibodies experienced no functional changes as compared to sham mice with no treatments at all (data not shown). M522 administration improved wall thickening at 10 weeks pi and reduced wall thickness, but did not significantly reduce end diastolic and systolic volumes, as compared to control. A t baseline, wall thickening was 91% but at 10 weeks p i , wal l thickening in control mice decreased to 46% while in the M522 animals, wall thickening declined only slightly to 74%, as shown in Figure 5.3A. L V P W thickness at baseline was 0.07 cm and 0.12 cm, in diastole and systole respectively, as shown in Figure 5.3B. Control wall thicknesses increased during both diastole and systole (0.10 and 0.14 cm, respectively) as compared to baseline at 10 weeks pi , while M522 wall thicknesses had returned towards baseline (0.08 cm in diastole and 0.13 cm in systole). Figure 5.3B demonstrates that at baseline, E D V and E S V were 0.026 and 0.007 m L respectively, however, at 10 weeks p i , E D V and E S V were subsequently increased in both M522 treated (0.033 and 0.01 m L , / ? « 0 . 0 0 1 , respectively) and control (0.038 and 0.012 m L , p « 0 . 0 0 1 ) groups as cardiac expansion and remodeling takes place. A trend in decreased E D V and E S V was observed in the M522 group as compared to controls (p=0.\). Systolic function, as measured by E F was reduced to 66% in both groups at week 10 as compared to 75% at baseline (not shown). Figure 5.2: Echocardiography analysis of cardiac function in mice following C V B 3 -infection. (A) Representative H & E sections of myocardium at 3, 9, and 30 days pi . Below are representative short axis ultrasound images taken at the level o f the papillary muscle and the tracings of the myocardial walls and chambers. Echocardiography was performed on days 3, 9, and 30 pi . Measurements for (B) L V P W thickening, (C) L V P W thickness, (D) L V E F , and (E) end diastolic and systolic volumes were recorded. Data are mean ± S.D., versus baseline. Scale bar denotes lOOpm. N=4 mice per timepoint. % p<0.02. 186 "3 B F - g r f- 4£^l^^m • * ; ': | 1 ' * , , i » & * i * % 1 ^ " #» . IL x : * ' t : • • • -X" ^—~—•— : ^ A * LVPW thickening D Bsln 3 9 30 Days Post-Infection Ejection Fraction ^ 60 9i LVPW Thickness Bsln 30 3 9 Days Post-Infection I Diastolic CH Systolic End Diastolic and Systolic Volumes 0.04 0.02 % % l I • T • T Bsln 30 Days Post-Infection Days Post-Infection 187 Figure 5.3: Echocardiography analysis of cardiac function following anti-4-lBBL treatment in CVB3-infected mice. (A) LV wall thickening, (B) wall thicknesses, and (C) ventricular volumes were measured at lOweeks pi . Data are mean ± S.D., baseline vs. M522 and control. #/?<0.005, ¥/?<0.05. A 120 | 80 40 LVPW Thickening 1 ] Baseline | M522 ] Control Bsln Week 10 B LVPW Thickness in Diastole and Systole c a se se 3. o 12 H ea Bsln Week 10 End Diastolic and Systolic Volumes ] Baseline LVPWd H Baseline LVPWs | M522 LVPWd Hj M522 LVPWs I I Control LVPWd 1 Control LVPWs I | Baseline EDV H Baseline ESV | M522 EDV H M522 ESV I I Control EDV 1 Control ESV Bsln Week 10 188 5.3.3 Histological Examination There was no significant difference in mortality rate between M522 treated and control mice. A s shown in Figure 5.4A, M522-treated animals exhibited significantly less myocardial injury than control animals (average scores 0 vs. 1.3,/?<0.05, respectively). The M522 group showed little or no cardiac injury while controls experienced greater damage, as evidenced by small diffuse lesions and scars (arrows), in their hearts. Some control animals experienced considerable histological injuries, with multiple large scars and calcific lesions. Cardiac sections were also subjected to picrosirius red staining for visualization of collagen amount and architecture, which was quantitated using Image-Pro Plus. Figure 5.4B shows that control hearts stained with picrosirius red revealed a marked increase in collagen deposition as well as soft tissue distortion as compared to M522 treated myocardium (4.8% vs. 3.3% collagen volume fraction, /?<0.05, respectively). Increased perivascular, replacement, and reactive fibrosis were also observed in control hearts while the M522 group demonstrated little fibrotic alterations. Such findings correlate with the histological grades which indicate that control hearts had more damage and lesions. During this late phase, most lesions were l ikely not active but had evolved into collagenous replacement scars and reactive fibrosis. 189 Figure 5.4: Histological analysis of morbidity and fibrosis in CVB3-infected mice following anti-4-lBBL treatment. (A) Representative H & E myocardial sections of M522 and control hearts with the corresponding histological scores. (B) Picrosirius staining for total collagen in M522-treated and control hearts and quantitation of collagen volume fraction as measured using the Image-Pro Plus® program. Scale bars=50pm. Data are mean ± S .D,¥ /?<0 .05 . M522 C O N T R O L in «** o — u <u u o o C/2 "3 ju 'So o "© X 2.5 1.5 0.5 M522 CONTROL 10 Weeks Post-infection B M522 C O N T R O L e u OS 0) S 3 > a v CD S o U M522 CONTROL 10 Weeks Post-infection 1 9 0 5.3.4 Protease Expression To determine i f proteases are involved in the differential remodeling between the M522-treated and control animal hearts, we used R T - P C R to compare the transcriptional levels of M M P - 2 , M M P - 9 , and M M P - 1 2 . These enzymes were demonstrated in previous chapters to be involved in acute myocarditis and may play a role in longterm remodeling [17]. We found that neither expression of M M P - 2 nor M M P - 9 transcripts was significantly altered between the two groups, as shown in Figure 5.5. M M P - 2 transcript was present abundantly in all samples whereas M M P - 9 was made minimally by both groups. Interestingly, M M P - 1 2 , or metalloelastase, was also expressed abundantly by both groups but the control group had increased expression of this transcript as compared to M522-treated animals, /?<0.05. 191 Figure 5.5: PCR analysis of MMPs-2, -9, and -12 in CVB3-infected mice following anti-4-1BBL treatment. (A) A representative blot showing semi-quantitative R T - P C R assessment for M M P - 2 , M M P - 9 , and M M P - 1 2 transcription between M522 and control mouse hearts. (B) Densitometric quantitation of the P C R bands. Data are mean ± SD, ¥ ^<0.05. M522 CONTROL M 5 2 2 C O N T R O L 5.3.5 Immunohistochemical Staining for Immune Cells To compare the degree of immune cell infiltration in M522-treated and control animals after C V B 3 infection, we stained for CD45 and C D 3 . A t 10 weeks p i , active myocarditis has largely subsided but various stimuli, such as persistent viral infection or autoimmunity, may cause ongoing low grade inflammation. Consistent with the histological 192 results, we found that control animals exhibited increased immunopositivity for CD45 , indicating more leukocytes infiltration than M522-treated animals (Figure 5.6A, arrows). Although differential CD45 infiltration did not reach statistical significance, a trend toward decreased CD45 immunopositivity in the M522-treatment group as compared to control hearts was observed (Figure 5.6B, p=0.0%). O f these cells, the majority were T-cells as shown by staining for C D 3 (Figure 5.6A) and the increase in this subset of lymphocytes is statistically significant (p<0.05). Due to the scarcity of lesions and scars within the M522-treated animals, most of the leukocytes were diffuse and dispersed amongst cardiomyocytes. In control animals, the number and size of lesions were increased and I detected extensive immunopositivity for both CD45 and C D 3 within and around these lesions (Figure 5.6A, arrows). 193 Figure 5.6: Immunohistochemistry analysis of CD45 and T-cells in CVB3-infected mice following anti-4-lBBL treatment. (A) Representative myocardial sections showing immunostaining for CD45 and CD3 in M522-treated and control hearts at 10 weeks pi . Staining is in brown (arrows). Quantitation of the immunoreactivity is represented in (B). Scale bars-50 um. Data are mean ± SD, ¥/?<0.05. A M 5 2 2 C O N T R O L M 5 2 2 C O N T R O L M 5 2 2 C O N T R O L 194 5.4 Conclusions and Discussion In this study, I treated CVB3-infected mice with neutralizing antibody or control non-blocking antibody for up to 10 weeks beginning from 2 weeks pi . A s such, I studied the chronic phase of this disease in a clinically-relevent model. I began treatment at day 15 to determine the role of the 4 - I B B pathway and T-cell regulation on cardiac function and this treatment regimen is relevant clinically as patients rarely present during a fulminant immune response. I show significant improvements in cardiac function after blocking 4 - 1 B B L interaction with its receptor, as measured by an increase in stroke volume, maintenance o f L V P W thickness, and contractility. I also observed less fibrosis, cardiac tissue injury, inflammation, and remodeling within the hearts treated with blocking antibody. Previous observations have shown that 4 - 1 B B L is increased in cardiomyocytes after C V B 3 infection in mice and in cases of human dilated cardiomyopathy [13,15]. In this study, I further show that both 4 - 1 B B L and 4 - I B B are increased in cardiomyocytes after virus infection. The pathogenesis of myocarditis is complex and often D C M and heart failure are the endpoints. Early virus-directed damage to the heart is through cytolysis, via both necrotic and apoptotic mechanisms, or "silent" infection of cells, which modulates both the behaviour of resident cardiac cells and the immune infiltrates. Even after the immune cells have entered the heart and cleared the virus, often irreparable damage has occurred through extensive myocyte death. The heart w i l l try to salvage structure and function through reparative fibrosis in areas o f myocyte dropout and this sets the stage for continual extracellular remodeling. If persistent viral infection or autoimmunity occurs, ongoing inflammation w i l l further threaten the viability of the heart. 195 We studied the 4 - I B B pathway in experimental myocarditis as it has previously been implicated in inflammation and apoptosis. 4 - I B B is expressed on CD8+ T-cells upon ligation with its ligand, which stimulates proliferation and activation o f these cells, thus aiding in tumour and virus clearance [18,19]. In vitro, stimulation of the 4 - I B B receptor activates both CD8+ and CD4+ T-lymphocytes but in vivo, the response is skewed towards stimulation o f CD8+ cells and abatement of CD4+ T-cells [20]. This pathway has important roles in influenza, herpes and H I V infections. Studies have shown that agonistic ant i -4- lBB antibodies improve anti-viral mechanisms exhibited predominantly by increased proliferation of CD8+ T-cells. The authors suggested that activation of the 4-1BB pathway upregulated INF-y secretion, increased proliferation of CD8+ T-cells, inhibited activation-induced cell death of these cells, and expanded memory T-cell population to protect against future infections [19,21]. However in our disease model, the immune response likely has both host protective and detrimental effects. During acute myocarditis, the immune response is crucial in clearing the virus and instigating the healing process, as suggested in Chapter I V . However, protracted inflammation is damaging to the myocardium. Many reports have shown that persistent virus infection can lead to immune-mediated attack on both infected and non-infected cardiomyocytes [22]. Autoimmunity may also play a role involving both CD8+ T-cells and auto-antibodies [22]. Therefore, this study was designed to target the late chronic phase of the disease, when active viral replication and inflammation have abated but ongoing low grade inflammation may still occur. B y treating CVB3-infected mice with antagonist a n t i - 4 - l B B L antibody to block activation o f this pathway, the detrimental effects o f T-cells are circumvented. I report here that immune cells, in particular T-cells, were indeed decreased in a n t i - 4 - l B B L treated animals. However, I did not investigate the 196 proportions of CD8+, CD4+ and yS-TCR cell subsets in this study and this knowledge can clarify the roles of each T-cell subtype in this model. Blockage of the 4 - I B B pathway during the late stage of the disease may decrease the activity of the detrimental CD8+ cells while sustaining the survival of CD4+ T-cells, thus improving the disease. The 4 - 1 B B / 4 - 1 B B L pathway is multifunctional. The 4 - I B B pathway induces apoptosis in several cell types but the mechanisms are unclear [4,11,12]. Although 4 - I B B receptor belongs to the T N F receptor superfamily, it does not have any death domains, such as Fas (CD95/APO-1) receptor, a characteristic element of the "extrinsic" apoptotic pathway. Upon binding to their ligand, these cell receptors aggregate into a death-inducing signaling complex (DISC) which also involves adaptor proteins, such as caspase 8. Activation o f such may lead to downstream activation of caspase 3 or Bcl-2 proteins [23-25]. Previous observations (unpublished data) have shown that 4 - I B B receptor can be induced on cardiomyocytes under ischemic and cytotoxic conditions. Inhibition o f activation of this pathway by using neutralizing an t i -4 - lBBL antibody improved disease after myocardial infarction and doxorubicin-induced cardiomyopathy, possibly by downregulation o f cardiomyocyte apoptosis via inhibition of mitochondrial dysfunction. This pathway involves induction of pro-apoptotic Bcl-2 proteins or creation o f transition pores in the mitochondria inner membrane, leading to leakage of cytochrome c activation of downstream caspases. Our laboratory has previously shown that mitochondrial disruption is a major cause of apoptosis during coxsackievirus infection and the virus often manipulates the host cell 's apoptotic pathways to maximize replication and dissemination [26,27]. Therefore, I speculate that 4-1BB may operate in a mitochondria-dependent apoptotic pathway. T U N E L staining and caspase 3 cleavage assays did not show any differences in treatment and control tissues (data 197 not shown). Vi ra l myocytolysis primarily occurs during acute myocarditis, therefore at week 10 pi , it may not have been detectable. Our results show that inhibition of 4 - I B B activation after C V B 3 infection contributed to positive cardiac remodeling. Picrosirius red staining demonstrated a reduction in lesions and scars within M522-treated mouse hearts. To further elucidate the mechanism for this phenomenon, we looked at the expression of M M P s . I have previously shown that the gelatinases M M P - 2 and M M P - 9 as well as the metalloelastase M M P - 1 2 were increased during acute myocarditis and remained upregulated at 30 days pi [17]. I hypothesized that these proteases regulate the immune response by modulating the activity and expression o f cytokines as well as in matrix remodeling. In this study, I found that M M P - 2 and M M P - 1 2 remained increased at 10 weeks pi in both groups while M M P - 9 , which we previously found to be predominantly secreted by immune cells, had returned to basal levels. However, metalloelastase was expressed substantially higher in the control group than the M522-treated group, suggesting that this enzyme may play a major role in negative matrix remodeling and disease severity. This correlated to an increase in both CD45 and C D 3 cells in control hearts. Even though immune cells are not the predominant source o f M M P - 1 2 , they produce many cytokines and other modulators of M M P s which induce resident cardiac cells to express M M P s [28]. Therefore, a decrease in inflammation means less cytokines to stimulate the expression of M M P - 1 2 . Results from Chapter III show that C V B 3 infection o f M M P 1 2 K O mice resulted in multi-organ injuries and dysfunctions, including increased viral titer and histological damage to the heart, which suggests that during the early phase o f infection, M M P - 1 2 is protective but longterm upregulation o f this enzyme may have detrimental effects. 198 In summary, this study demonstrated that both 4 - I B B and 4 - 1 B B L are expressed by cardiomyocytes during the viremic phase of myocarditis, day 9 p i , and also chronically during the reclamative phase, week 10 pi . Blockade of this pathway reduced histological cardiac damage, diminished inflammatory infiltrate, and enhanced cardiac function. Our data suggests that 4 - I B B activation may contribute to the chronic cardiac dysfunction through longterm T-cell activation following C V B 3 infection. In conclusion, I found that a neutralizing a n t i - 4 - l B B L antibody may be a novel therapeutic agent to prevent the chronic cardiac sequelae after CVB3-induced myocarditis. Most importantly, I have demonstrated that administration of a neutralizing antibody after the viremic phase o f infection had beneficial effects on chronic cardiac function. Modulation of 4 - I B B pathway may be a therapeutic target for human myocarditis-induced heart failure. 199 5.5 References 1. Watts T H . T N F / T N F R family members in costimulation of T cell responses. Annu Rev Immunol 2005;23:23-68. 2. K i m JO, K i m H W , Baek K M , Kang C Y . 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K i m Y J , Mantel P L , June C H , K i m S H , K w o n B S . 4 - I B B costimulation promotes human T cell adhesion to fibronectin. Cel l Immunol 1999;192:13-23. 8. Myers L M , Vel la A T . Interfacing T-cell effector and regulatory function through CD137 (4-1BB) co-stimulation. Trends Immunol 2005;26:440-446. 9. K i m J, Choi W S , L a S, Suh J H , K i m B S , Cho H R , et al. Stimulation with 4 - I B B (CD 13 7) inhibits chronic graft-versus-host disease by inducing activation-induced cell death of donor CD4+ T cells. Blood 2005;105:2206-2213. 10. K w o n B , Lee H W , K w o n B S . New insights into the role of 4-1BB in immune responses: beyond CD8+ T cells. Trends Immunol 2002;23:378-380. 11. Heinisch IV, Daigle I, Knopfl i B , Simon H U . C D 137 activation abrogates granulocyte-macrophage colony-stimulating factor-mediated anti-apoptosis in neutrophils. Eur J Immunol 2000;30:3441-3446. 12. Miche l J, Pauly S, Langstein J, Krammer P H , Schwarz H . 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Expression of tumor necrosis factor ligand superfamily costimulatory molecules C D 2 7 L , C D 3 0 L , O X 4 0 L and 4 - 1 B B L in the heart of patients with acute myocarditis and dilated cardiomyopathy. Cardiovasc Pathol 2002; 11:166-70. 16. Yanagawa B , Spiller O B , Proctor D G , Choy J, Luo H , Zhang H M , et al. Soluble recombinant coxsackievirus and adenovirus receptor abrogates coxsackievirus b3-mediated pancreatitis and myocarditis in mice. J Infect Dis 2004;189:1431-1439. 17. Cheung C, Luo H , Yanagawa B , Leong H S , Samarasekera D , L a i JC , et al. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in coxsackievirus-induced myocarditis. Cardiovasc Pathol 2006;15:63-74. 18. Zhang H , Knutson K L , Hellstrom K E , Disis M L , Hellstrom I. Antitumor efficacy o f CD137 ligation is maximized by the use of a C D 137 single-chain Fv-expressing whole-cell tumor vaccine compared with CD137-specific monoclonal antibody infusion. M o l Cancer Ther 2006;5:149-155. 19. K i m Y H , Seo S K , Choi B K , Kang W J , K i m C H , Lee S K , et al. 4 - I B B costimulation enhances H S V - 1 -specific CD8+ T cell responses by the induction of CD1 lc+CD8+ T cells. Ce l l Immunol 2005;238:76-86. 20. Vinay D S , Cha K , K w o n B S . Dual immunoregulatory pathways of 4-1BB signaling. J M o l M e d 2006;84:726-736. 21. DeBenedette M A , Wen T, Bachmann M F , Ohashi PS, Barber B H , Stocking K L , et al. Analysis of 4 - I B B ligand (4-lBBL)-deficient mice and of mice lacking both 4 - 1 B B L and CD28 reveals a role for 4 - 1 B B L in skin allograft rejection and in the cytotoxic T cell response to influenza virus. J Immunol 1999;163:4833-4841. 22. Huber S A , Born W , O'Brien R. Dual functions o f murine gammadelta cells in inflammation and autoimmunity in coxsackievirus B3-induced myocarditis: role of Vgammal+ and Vgamma4+ cells. Microbes Infect 2005;7:537-543. 23. Kroemer G , Martin SJ. Caspase-independent cell death. Nat M e d 2005; 11:725-730. 24. Green D R , Kroemer G . The pathophysiology of mitochondrial cell death. Science 2004;305:626-629. 201 25. Granville D J , Carthy C M , Yang D , Hunt D W , McManus B M . Interaction of viral proteins with host cell death machinery. Cel l Death Differ 1998;5:653-659. 26. Carthy C M , Yanagawa B , Luo H , Granville D J , Yang D , Cheung P, et al. Bcl -2 and B c l - x L overexpression inhibits cytochrome c release, activation of multiple caspases, and virus release following coxsackievirus B3 infection. Virology 2003;313:147-157. 27. Zhang H M , Yanagawa B , Cheung P, Luo H , Yuan J, Chau D , et al. Nip21 gene expression reduces coxsackievirus B3 replication by promoting apoptotic cell death via a mitochondria-dependent pathway. Circ Res 2002;90:1251-1258. 28. Shimizu K , Shichiri M , Libby P, Lee R T , Mitchell R N . Th2-predominant inflammation and blockade of IFN-gamma signaling induce aneurysms in allografted aortas. J C l i n Invest 2004;114:300-308. 202 CHAPTER VI: CONCLUSIONS AND FUTURE DIRECTIONS In this dissertation, I presented the data I collected in the last 5.5 years, working under the direction of Dr. McManus in dissection of the roles of M M P s in CVB3-induced myocarditis. First I investigated the expression levels of select M M P s and all four TIMPs . M M P - 2 , M M P - 9 , M M P - 1 2 , and M T 1 - M M P were all increased while M M P - 8 was downregulated during the inflammatory and chronic phases of myocarditis. This was associated with corresponding decreases in TIMP-3 and TIMP-4 . The most unique and novel finding from this study was the detection of M M P - 1 2 in cardiomyocytes and its subsequent upregulation following C V B 3 infection. In this study, I did not investigate the source of the M M P s and TIMPs , via techniques such as in situ hybridization, which would shed light on the significance o f these alterations and the underlying mechanisms. In order to examine these mechanisms, I used genetically modified mice deficient in M M P - 1 2 , M M P - 8 , or M M P - 9 and infected them with C V B 3 . Other M M P s , such as M M P - 2 , M T 1 - M M P , and M M P - 1 3 were also examined in this dissertation but I chose to further investigate M M P - 1 2 , M M P - 8 , and M M P - 9 because of their direct association with inflammation and cytokine processing. Interestingly, CVB3-infected M M P - 1 2 knockout mice exhibited the most severe infection, such that these mice did not survive past 3 days pi . The viral infection was not exclusive to the myocardium but resulted in multi-organ injuries. The basis for this novel protective function of M M P - 1 2 is unclear and warrants further investigations into the mechanisms by performing more in vitro studies. It is unlikely that M M P - 1 2 functions in maladaptive remodeling during this early stage of the disease and I reported little changes in E C M architecture in virus-infected M M P 1 2 K O heart, pancreas, 203 spleen, and liver. In the M M P 1 2 K 0 liver, degradation of the collagen network was observed but this occurrence is probably a byproduct of excessive cytolysis o f hepatocytes rather than M M P dysregulation. Investigations into the M M P and T I M P expression profiles in M M P 1 2 K O mice would clarify this issue. The observation that M M P - 1 2 was produced by cardiomyocytes and possibly other cell types suggests that this enzyme may play a role in signalling rather than E C M degradation. It is of special interest to elucidate how virus infection can upregulate M M P - 1 2 in infected cells. M M P - 9 was also shown to play a protective role in myocarditis, as evidenced by increased cardiac injury, immune cell infiltration (T-cells), and cytokine induction. The differential activation o f the immune response could be a direct manifestation of M M P - 9 deficiency, since this protease has many immunomodulatory functions, or an indirect effect of ineffective virus elimination leading to a delayed, expanded immune response. To clarify this, it is necessary to examine the myocardial condition of CVB3-infected M M P 9 K O mice at earlier timepoints, such as day 3 pi . It appears that M M P - 9 and M M P - 1 2 have different functions during viral infection, since I reported that M M P - 9 was predominately expressed by leukocytes and M M P - 1 2 was co-localized to the myocardium. Further, viral infection in mice deficient in either one of these proteases exacerbated the disease while infection in M M P 8 K O showed little alterations as compared to controls. However, one key difference is that in the M M P 8 K O mice, there was a significant decrease in T-cell infiltration. This is particularly interesting since both M M P - 8 and M M P - 9 have been shown to have similar substrates, they are both produced and stored within neutrophilic granules, and they are important for P M N effector functions. In order to determine the significance of M M P - 8 in viral myocarditis, it is necessary to examine 204 virus infection in M M P 8 K O mice during chronic disease. Since M M P - 8 is a major collagenase and T-cells were downregulated in the M M P 8 K O mice, it is possible that chronic myocarditis would be ameliorated within the M M P 8 K O mice due to decreases in maladaptive remodeling and persistent inflammation. These results suggest that M M P - 1 2 and M M P - 9 have protective functions during early infection while M M P - 8 may have detrimental functions especially during the chronic phase. Many studies have implicated disfavourable roles of T-cells in C V B 3 myocarditis and in our K O models, T-cell infiltration was dysregulated and may contribute to pathogenesis of disease. T-lymphocytes may have both beneficial and detrimental functions in myocarditis, depending on the time course of infection. Early in the disease, T-cells aid in virus elimination and regulation of wound repair but persistent, chronic infiltration w i l l result in increased damage to the heart. Therefore, I inhibited T-cell activation specifically during the chronic phase of myocarditis when the detrimental effects of T-cells would have occurred. This treatment scheme is clinically relevant to patients presenting with this disease, since the onset of myocarditis is not clear and diagnosis is often delayed. The 4 - I B B pathway has been shown in previous reports to be particularly important in T-cell activation in myocarditis. I used neutralizing antibodies against this pathway and showed a significant improvement in cardiac damage, L V function, immune infiltration, and matrix remodeling. Notably, M M P - 1 2 was decreased after treatment, which suggests that this enzyme may function in a two part process: early infection requires M M P - 1 2 to fend off the virus while this protease may have unfavourable immunomodulatory effects during late stage myocarditis. 205 In regards to clinical significance, detection of M M P s and TIMPs in endomyocardial biopsy samples by either immunohistochemistry or P C R may be help in determining whether there is increased expression of certain M M P s . M M P or T-cell suppression should not begin during acute myocarditis because of their protective properties. Anti-viral treatment should be administered at the onset of clinical presentation with histological confirmation of acute myocarditis. Our laboratory is also exploring novel therapeutic agents against coxsackieviral infection. Recent investigations in the utilization of gene therapy, including small interfering R N A , antisense oligodeoxynucleotides (AS-ODNs) , and phosphorodiamidate morpholino oligomers, for treatment of C V B 3 infection have shown that specific targeting of the viral genome is able to decrease viral replication and dissemination, host cell cytolysis, and cardiac tissue injury. These results demonstrate a great potential for further development of gene therapy against viral myocarditis as well as other diseases caused by C V B 3 infection. During chronic myocarditis, ongoing inflammation and maladaptive remodeling are the driving forces behind continual myocardial injury. If increased M M P - 1 2 expression is detected during subsequent biopsies, even with no indication of chronic inflammation, then M M P - 1 2 inhibition may be helpful i n decreasing maladaptive remodeling. Further, increased M M P - 8 may be associated with increased T-cell infiltration, which may be detrimental particularly during chronic myocarditis; therefore, M M P and 4-IBB inhibition may improve both the structural as well as functional integrity of the infected heart. In this dissertation, the significance of M M P - 1 2 , M M P - 9 , and T-cells in myocarditis are shown in the early and chronic stages of myocarditis. However, the exact mechanisms by which they operate are still unknown and require further investigations. These elements 206 function in a highly complex network and the timing of their activation must be properly regulated in order for normal virus and cardiac repair resolution to occur. Figure 7.1: Illustration of hypothesized MMP mechanisms during the three phases of myocarditis. During the acute phase of C V B 3 infection, infected cells upregulate the synthesis and secretion of M M P - 1 2 , which may play a protective role since ablation o f this enzyme in C V B 3 -infected mice greatly increases disease severity. M M P - 1 2 may play multiple roles during this early phase of infection: 1) activation and recruitment o f innate cells, such as phagocytes, which aid in clearance of the virus; 2) cleavage of virus receptors or associated signaling molecules in order to circumvent viral uptake into cells; 3) activation o f the innate immune program through regulation of interferons and Toll- l ike receptors; and 4) inhibition of apoptosis, which reduces viable tissue and increases injury to the organ. During the inflammatory phase of the disease, infected cardiomyocytes increase secretion of a variety o f cytokines to recruit and activate phagocytes, which secrete M M P - 9 . This enzyme can activate and inactivate various cytokines, growth factors, and cell receptors through proteolytic cleavage to modulate the immune response, in particular the T-cells, as evidenced by increased T-cell infiltration in M M P 9 K O mice. Infected cardiomyocytes also upregulate 4 - I B B , leading to activation and recruitment of T-cells, which aid in viral clearance but may also contribute to autoimmunity and propagation of tissue damage. Prolonged activation of the 4 - I B B pathway may lead to chronic myocarditis, by maintaining inflammation and T-cell infiltration. This continual inflammation may signal cardiomyocytes to secrete M M P - 1 2 in order to repair and remodel the injured myocardium. 207 CARI DAF Myocyte / I \ ACUTE INFLAMMATORY CHRONIC APPENDIX A.l List of Publications, Abstracts, and Presentations Peer-Reviewed Publications 1. Cheung C, Deisher T, Yanagawa B , Luo H , Bonigut S, Samra A , Zhao H , Walker E , McManus B . Neutralizing an t i -4 - lBBL treatment improves cardiac function in viral myocarditis. In press (Lab Invest). 2. Cheung C, Luo H , Yanagawa B , Leong H , Samarasekera D , L a i J, Suarez A , Zhang J, McManus B . Matrix metalloproteinases and tissue inhibitors o f metalloproteinases in coxsackievirus-induced myocarditis. Cardiovasc Pathol, 2006 Mar-Apr;15(2): 63-74. 3. S i X , Luo H , Morgan A , Zhang J, Wong J, Yuan J, Esfandiarei M , Gao G , Cheung C, McManus B M . Stress-activated protein kinases are involved in coxsackievirus b3 viral progeny release. J Virol, 2005 Nov; 79(22): 13875-81. 4. S i X , McManus B M , Zhang J, Yuan J, Cheung C, Esfandiarei M , Suarez A , Morgan A , Luo H . Pyrrolidine dithiocarbamate reduces coxsackievirus B3 replication through inhibition of the ubiquitin-proteasome pathway. J Virol, 2005; 79(13):8014-23. 5. Yuan J, Cheung P K , Zhang H , Chau D , Yanagawa B , Cheung C, Luo H , Wang Y , Suarez A , McManus B M , Yang D . A phosphorothioate antisense oligodeoxynucleotide specifically inhibits coxsackievirus B3 replication in cardiomyocytes and mouse hearts. Lab Invest, 2004; 84(6):703-l. 6. Carthy C, Yanagawa B , Luo H , Granville D J , Yang D C , Cheung P, Cheung C, Esfandiarei M , Rudin C M , Thompson C B , Hunt D W C , McManus B . M . Bcl -2 and bcl-xl overexpression inhibits cytochrome C release, activation of multiple caspases and virus release following coxsackievirus B3 infection. Virology, 2003; 313(l):147-57. 7. Luo H , Zhang JC, Cheung C, Suarez A , McManus B M , Yang D C . Proteasome inhibition reduces coxsackievirus B3 replication in murine cardiomyocytes. Am J Pathol, 2003; 163(2):381-5. Reviews and Chapters 1. Rahmani M , Wong B W , A n g L , Cheung C, Carthy J M , Walinski H , McManus B M . Versican: signaling to transcriptional control pathways: Can J Physiol Pharmacol. 2006 Jan; 84(l):77-92. 209 2. Yang D C , Cheung P, Yanagawa B , Chau D , Cheung C, McManus B M . (2002) Picornaviral proteases in viral replication and pathogenesis. Current Topics in Virology. India: Research Trends. Manuscripts Submitted and In Preparation 1. Cheung C, Luo H , Yanagawa B , Leong H , Zhang J, Rahmani M , Walinski H , Walker E , McManus B . Ablation of M M P - 9 in CVB3-infected mice increases severity of myocarditis. Submitted to Circulation. 2. Cheung C, Marchant D , Luo H , Yanagawa B , Zhang J, Yuan J, Rahmani M , Walinski H , Walker E , McManus B . M M P - 1 2 deficiency in coxsackievirus-infected mice exacerbates myocarditis, hepatitis, and pancreatis. In preparation. 3. Wang X , Bonigut S, Alford K , Cheung C, Deisher T. Inhibition of the tumor necrosis factor receptor family member, 4 - I B B , improves cardiac function in cytotoxic and ischemic mouse models. Submitted to Circ Res. 4. Yanagawa B , Luo H , Cheung C, Deisher T A , Yuan J, Hollander Z , Wong B , Kapoun A M , Bonigut S, Yang D , Schreiner G F , N g R, McManus B M . Alterations in host survival and remodeling gene expression in CVB3-infected mouse hearts. Submitted to Life Sci. 5. Gao G , Zhang J, Si X , Wong J, Cheung C, Luo H . Protection o f coxsackievirus-induced murine myocarditis by proteasome inhibition. Submitted to Am J Pathol. 6. Walinski H , Pate G , Leong H , Rahmani M , Cheung C, McManus B , Podor T. Vitronectin binds to desmin at sites of acute myocardial infarction and reduces cardiomyocyte contractility. Submitted to PNAS. Abstracts and Oral Presentations 1. Yanagawa B , Luo H , Schreiner G , Yuan J, Cheung C, Zhang M , Cheung P, Yang D C , Bonigut S, Deisher T, McManus B M . Gene profiling in CVB3-Infected mouse hearts. Horizons in Heart Failure Conference. San Diego, C A , December 2002. 2. Yanagawa B , Luo H , Yuan J, Schreiner G F , Cheung C, Wong B , Zhang M , Cheung P, Yang D C , McManus B M . Therapeutic horizons in heart failure: gene profiling in cvb3-infected mouse hearts. L a Jolla, C A , December 2002. 3. Cheung C, Yanagawa B , Luo H , La i J C K , Zhang J, McManus B M . Expression and activation of matrix metalloproteinases-2 and -9 during coxsackievirus infection of murine hearts. United States and Canadian Association of Pathologists Conference. Washington D C , March 2003. 210 4. Cheung C, Luo H , Zhang J, Sirianni F, Yuan J, Zhang M , Yanagawa B , Walker D C , McManus B M . Characterization of cultured murine cardiomyocytes after coxsackievirus infection. Experimental Biology ( F A S E B ) . San Diego, C A , A p r i l 2003. 5. Cheung C, Yanagawa B , Luo H , Suarez A , L a i J C K , Zhang J, McManus B M . Expression and activation of matrix metalloproteinases during coxsackievirus infection of mouse hearts. Presented at Molecular Mechanisms of Growth, Death, and Regeneration in the Myocardium: Basic Biology and Insights into Ischemic Heart Disease and Heart Failure (AHA). Snowbird, Utah, August 2003. 6. Cheung C, Yanagawa B , Luo H , Suarez A , La i J C K , Zhang J, Sirianni F, McManus B M . Expression and activation of M M P s and TIMPs following coxsackieivirus-induced myocarditis. United States and Canadian Association of Pathologists Conference, Vancouver, B C , March 2004. 7. Si X , Zhang J, Esfandiarei M , Yuan J, Cheung C, Luo H , McManus B . Pyrrolidine dithiocarbamate and zinc synergistically inhibit coxsackievirus b3 replication in cultured cells. C I H R National Research Forum for Young Investigators in Circulatory and Respiratory Health. Winnipeg, 2004. Exp Clin Cardiol. 2004; 9(1): 80. 8. Cheung C, Yanagawa B , Luo H , Suarez A , La i J C K , Zhang J, Sirianni F, McManus B M . Expression and activation of M M P s and TIMPs following coxsackieivirus-induced myocarditis. Experimental Biology. Washington D C , April 2004. Invited oral presentation. 9. Cheung C, Yanagawa B , Luo H , Zhang J, McManus B M . Cardiomyocytes upregulate matrix metalloproteinases following coxsackievirus infection. Heart Failure Society of American Conference. Toronto, O N , September 2004. 10. McManus B M , Spiller B , Deischer T, Luo H , Cheung C, Triche T, Yanagawa B . From expression profiling to biological validation in coxsackievirus infections: how far to leap, when, and where? The X V I I I World Congress International Society for Heart Research. Brisbane, Australia, 2004. 1.1. Cheung C , Yanagawa B , Luo H , Zhang J, McManus B M . Cardiomyocytes upregulate matrix metalloproteinases following coxsackievirus infection. The 2 n d Annual C I H R National Research Forum for Young Investigators in Circulatory and Respiratory Health. Winnipeg, M B , A p r i l 2005. Exp Clin Cardiol, 2005. 12. Luo H , Si X , Gao G , Wong J, Wang Y , Zhang, Cheung C, McManus B M . Ubiquitin-mediated protein modification and degradation: a novel mechanism o f coxsackievirus infectivity. The 3 r d Annual C I H R National Research Forum for Young Investigators in Circulatory and Respiratory Health. Winnipeg. M a y 2006. Exp Clin Cardiol, 2006. 211 Awards 1. Dr. David Hardwick Studentship, University of British Columbia, Canada, Effective: 09/2001, Ending: 05/2002, Department of Pathology and Laboratory Medicine, $10,500 2. Doctoral Research Award - D E C L I N E D , Heart and Stroke Foundation o f Canada, Canada, Cardiovascular Pathology. 3. Doctoral Research Award, Canadian Institutes of Health Research, Canada, Effective: 04/2003, Ending: 04/2006, Cardiovascular Pathology, $63,000 4. Albert B and Mary Steiner Summer Research Award, Vancouver Foundation, Canada, Effective: 05/2004, Ending: 08/2004, Merit based award to those top 10% of their class, $650 2 1 2 

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