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Impact of diesel exhaust inhalation on the heart and blood vessels Bai, Ni 2011

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Impact of Diesel Exhaust Inhalation on the Heart and Blood Vessels  by  Ni Bai  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate Studies (Pharmacology and Therapeutics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  June, 2011  Ni Bai, 2011  i1  1  Abstract Numerous epidemiological studies have shown that exposure to ambient particulate matter (PM) is implicated in increased cardiovascular morbidity and mortality; however, the biological mechanisms are not fully understood. Diesel exhaust (DE) is the single biggest contributor to the urban ambient PM, and accounts for up to 90% of the total mass of fine particulate mass in ambient air of many major cities, such as London. In this dissertation, I evaluated the effects of DE inhalation at an environmentally relevant level in a mouse model of atherosclerosis, the apolipoprotein E deficient (apoE knockout) mouse. I hypothesized that exposure to DE causes progression of atherosclerosis and vascular dysfunction, which leads to cardiovascular morbidity and mortality. I used a morphometric analysis to determine the compositional changes in atherosclerotic plaques, and showed that DE inhalation increased lipid accumulation, foam cell formation and smooth muscle cell recruitment in plaques, whereby suggesting a progression of atherogenesis. The magnitude of DE deposition in the lung correlates with foam cell formation suggesting a strong link between DE inhalation and atherogenesis. Oxidative stress markers, including CD36 and nitrotyrosine, were all increased after exposure to DE, suggesting that reactive oxygen species played an important role in this vascular effect. In addition, I showed that exposure to DE up-regulated iNOS and COX2 expression at both protein and mRNA levels in blood vessel and heart tissue. A functional study of blood vessels showed no impairment of acetylcholine (ACh) relaxation, but the sodium nitroprusside-stimulated endothelium-independent relaxation was enhanced following DE exposure. This could be partly explained by an increase in soluble guanylate cyclase expression in blood vessels. However, there was attenuated phenylephrine (PE)-stimulated vasoconstriction induced by DE exposure. An increased iNOS-derived NO production and up-regulation of COX2 could contribute to this attenuated constriction. ii  In conclusion, I demonstrate that DE inhalation alters the composition of atherosclerotic plaque resulting in unstable plaques that are vulnerable to rupture. Oxidative stress and iNOS up-regulation contribute to these DE exposure-induced vascular effects. We postulate that compensatory effects are activated to minimize the deleterious impact of DE exposure on vascular function.  iii  Preface This dissertation is based on 4 manuscripts, resulting from collaboration between multiple researchers.  A version of Chapter 3 has been published by Atherosclerosis. This paper is coauthored with Ni Bai, Takashi Kido, Hisashi Suzuki, Grace Yang, Terrance J. Kavanagh, Joel D. Kaufman, Michael E. Rosenfeld, Cornelis van Breemen, Stephan F van Eeden. Ni Bai and Stephan van Eeden developed the experimental design and protocols. Ni Bai performed all animal experiments, part of the data analysis (except the immunostaining), and wrote the manuscript. Takashi Kido and Hisashi Suzuki helped with harvesting the tissues. Grace Yang helped with data analysis for immunostaining. Part of the work (DE exposure, sample collection and processing) was conducted in the labs of Drs. Terrance J. Kavanagh, Joel D. Kaufman, and Michael E. Rosenfeld.  A version of Chapter 4 has been accepted for publication by Toxicology and Applied Pharmacology. This paper is co-authored with Ni Bai, Takashi Kido, Hisashi Suzuki, Terrance J. Kavanagh, Joel D. Kaufman, Michael E. Rosenfeld, Cornelis van Breemen, Stephan F. van Eeden. Ni Bai and Stephan van Eeden developed the experimental design and protocols. Ni Bai performed all animal experiments, data analysis, and wrote the manuscript. Takashi Kido and Hisashi Suzuki helped with harvesting the tissues. Part of the work (DE exposure and vascular functional study) was conducted in the labs of Drs. Terrance J. Kavanagh, Joel D. Kaufman, and Michael E. Rosenfeld.  A version of Chapter 5 has been submitted to a peer-reviewed journal for publication. The paper is co-authored with Ni Bai, Erin M. Tranfield, Terrance J. Kavanagh, Joel D. iv  Kaufman, Michael E. Rosenfeld, Stephan F. van Eeden. Ni Bai and Stephan van Eeden developed the experimental design and protocols. Ni Bai performed all animal experiments, data analysis, and wrote the manuscript. Erin M. Tranfield helped with harvesting the tissues. Part of the work (DE exposure and vascular functional study) was conducted in the labs of Drs. Terrance J. Kavanagh, Joel D. Kaufman, and Michael E. Rosenfeld.  A version of Chapter 6 will be submitted to a peer-reviewed journal for publication. This paper is co-authored with Ni Bai, Hisashi Suzuki, Terrance J Kavanagh, Joel D Kaufman, Michael E. Rosenfeld, Stephan F. van Eeden. Ni Bai and Stephan van Eeden developed the experimental design and protocols. Ni Bai performed all animal experiments, data analysis, and wrote the manuscript. Hisashi Suzuki helped with harvest the tissues. Part of the work (DE exposure and vascular functional study) was conducted in the labs of Drs. Terrance J. Kavanagh, Joel D. Kaufman, and Michael E. Rosenfeld.  In addition to general research directions, Dr. Stephan van Eeden, as my supervisor, helped with his numerous suggestions in the course of enhancing the experimental design, data analysis, as well as editing all the manuscripts.  Animal procedures were approved by the Animal Care and Use Committee of the University of Washington (Protocol number: 2132-04).  v  Table of Contents Abstract…………………………………………………………………………………..….ii Preface…………………………………………………………………………………..…...iv Table of Contents……………………………………………………………………..…….vi List of Tables……………………………………………………………………………..…xi List of Figures………………………………………………………………………...…….xii List of Abbreviations and Acronyms…...……………………………………...……….....xv Acknowledgements…………………………………………………………………….…xvii Dedication………………………………………………………………………………...xviii CHAPTER 1: INTRODUCTION ··································································································· 1 1.1 Background ········································································································································· 1 1.1.1 Particulate Matter Air Pollution or PM10 ·············································································· 2 1.1.2 Epidemiological Evidence for Adverse Cardiovascular Effects Associated with Exposure to Particular Matter Air Pollution (PM10) ····································································· 7 1.1.2.1 Acute Cardiovascular Effects Induced by PM10 Exposure ······································· 7 1.1.2.2 Chronic Cardiovascular Effects Induced by PM10 Exposure···································· 9 1.1.3 Proposed Pathways for PM10–Induced Cardiovascular Effects ····································· 13 1.1.3.1 PM10-Induced Pulmonary and Systemic Inflammation ··········································· 14 1.1.3.1.1 Lung Inflammatory Response Induced by PM10 ··············································· 15 1.1.3.1.1.1 Alveolar Macrophage Response to PM10 Exposure ································· 15 1.1.3.1.1.2 Lung Epithelial Cell Response to PM10 Exposure ···································· 17 1.1.3.1.1.3 Alveolar Macrophage and Lung Epithelial Cell Interaction ··················· 17 1.1.3.1.2 Systemic Inflammatory Response Induced by PM10 Exposure······················ 19 1.1.3.1.2.1 Circulating Mediator Release Induced by PM10 Exposure ····················· 19 1.1.3.1.2.2 Acute Phase Response Induced by PM10 Exposure ·································· 20 1.1.3.1.2.3 Bone Marrow Stimulation Induced by PM10 Exposure ··························· 20 1.1.3.2 Extra-Pulmonary Translocation of PM10 ···································································· 22 1.1.4 PM10 and Atheroslcerosis······································································································· 23 1.1.4.1 Atherosclerosis ················································································································· 23 1.1.4.1.1 Pathogenesis of Atherosclerosis ··········································································· 23 1.1.4.1.2 Apolipoprotein E Knockout Mouse-a Mouse Model for Atherosclerosis ··· 25 1.1.5 Mediators that Contribute to the Development of Atherosclerosis Induced by Exposure to PM10 ······························································································································· 27 1.1.5.1 Endothelium and Nitric Oxide Regulation and Cardiovascular Dysfunction ····· 27 1.1.5.2 iNOS and Cardiovascular Dysfunction ······································································· 32 1.1.5.3 CD36 and Cardiovascular Dysfunction······································································· 34 1.1.5.4 Peroxynitrite and Cardiovascular Dysfunction ·························································· 35 1.1.5.5 PM10 Exposure and Oxidative Stress ··········································································· 36 1.1.5.6 Cyclooxygenase (COX) and Cardiovascular Dysfunction······································ 38 vi  1.2 Objective, Hypothesis, and Specific Aims ················································································· 41 1.2.1 PM10 and Cardiovascular Diseases ······················································································ 41 1.2.2 Shortcomings of Published Studies······················································································ 42 1.2.3 Diesel Exhaust (DE) ················································································································ 43 1.2.4 Objective, Rationales and Specific Aims············································································ 43 1.2.4.1 Rationale for Specific Aim 1 ························································································· 44 1.2.4.2 Rationale for Specific Aim 2 ························································································· 44 1.2.4.3 Rationale for Specific Aim 3 ························································································· 45 1.2.4.4 Rationale for Specific Aim 4 ························································································· 46 CHAPTER 2: METHODOLOGY ································································································ 47 2.1 Exposure Protocol and Experimental Animals ·········································································· 47 2.1.1 DE Exposure System ·············································································································· 47 2.1.2. Animals and Exposure Protocol ·························································································· 48 2.2 Sample Collection···························································································································· 48 2.3 Peripheral Band Cell Counts ········································································································· 49 2.4 Tissue Processing and Image Acquisition ·················································································· 50 2.5 Morphometric Analysis of the Lung Tissue and the Composition of Atherosclerotic Plaque ························································································································································ 50 2.5.1 Lung Tissue ······························································································································· 50 2.5.2 Atherosclerotic Lesions in Aortic Root ··············································································· 51 2.5.2.1 Lesion Size, Foam Cell and Cellularity ······································································ 51 2.5.2.2 Lipid Content of Plaque·································································································· 51 2.5.2.2 Collagen Content of Plaque ··························································································· 52 2.6 Vascular Function Study ·········································································· 52 2.7 Immunohistochemical Analysis of Macrophages (F4/80), iNOS, CD36, Nitrotyrosine, α-actin, COX1, COX2, eNOS, and sGC Expression ······································································ 53 2.8 Western Immunoblotting Analysis of iNOS Expression ························································· 54 2.9 ELISA of NF-κB (p65) Activity··································································································· 55 2.10 Assessment of Systemic Oxidative Stress ················································································ 55 2.11 Measurement of Urine 2,3-dinor-6-keto prostaglandin F1α ················································ 56 2.12 Measurement of Plasma Nitrite and Urine cGMP ·································································· 56 2.13 Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) of mRNA Expression of iNOS, NT, NFκB, COX1, COX2 and PGI2 in the Heart, and mRNA Expression of eNOS and sGCα in the Abdominal Aorta······························································· 57 2.14 Solutions and Chemicals ·············································································································· 57 vii  2.15 Statistical Analysis ························································································································ 57 CHAPTER 3: CHANGES IN ATHEROSCLEROTIC PLAQUES INDUCED BY INHALATION OF DIESEL EXHAUST ······················································································ 59 3.1 Introduction ······································································································································· 59 3.2 Methods and Materials ··················································································································· 61 3.2.1 Methods ····································································································································· 61 3.2.2 Statistical Analysis ·················································································································· 62 3.3 Results ················································································································································ 63 3.3.1 Plasma Lipid ····························································································································· 63 3.3.2 Alveolar Macrophages in the Lung Tissue ········································································· 63 3.3.3 Increased Circulating Band Cells and Positive Correlation with Lung Inflammation ································································································································································ 64 3.3.4 Compositional Changes in Atherosclerotic Plaque ·························································· 65 3.3.5 Increased Oxidative Stress in Atherosclerotic Plaque······················································ 70 3.3.6 Systemic Oxidative Stress······································································································ 74 3.4 Discussion ········································································································································· 74 3.4.1 Compositional Changes in Atherosclerotic Plaque ·························································· 76 3.4.2 Oxidative Stress and Compositional Changes in Atherosclerotic Plaque ···················· 78 3.5 Conclusions ······································································································································· 80 CHAPTER 4: EXPOSURE TO DIESEL EXHAUST UP-REGULATES INOS ACTIVITY IN APOE KNOCKOUT MICE ················································································ 81 4.1 Introduction ······································································································································· 81 4.2 Methods and Materials ··················································································································· 83 4.2.1 Methods ····································································································································· 83 4.2.2 Solutions and Chemicals ········································································································ 84 4.2.3 Statistical Analysis ·················································································································· 85 4.3 Results ················································································································································ 85 4.3.1 iNOS Activity and Expression in the Thoracic Aorta ······················································ 85 4.3.2 iNOS Expression in the Heart Tissue ·················································································· 89 4.3.3 Co-localization of iNOS with CD36 and Nitrotyrosine Expression ····························· 91 4.3.4 NF-κB (p65)-Mediated iNOS Expression ·········································································· 93 4.4 Discussion ········································································································································· 94 4.4.1 The Level of DE Exposure ···································································································· 95 4.4.2 DE Exposure and Up-regulation of iNOS ·········································································· 95 4.4.3 iNOS and Atherosclerosis ······································································································ 97  viii  CHAPTER 5: EXPOSURE TO DIESEL EXHAUST CAUSES UP-REGULATION OF COX2 ACTIVITY IN APOE KNOCKOUT MICE ··························································· 99 5.1 Introduction ······································································································································· 99 5.2 Methods and Materials ················································································································· 101 5.2.1 Methods ··································································································································· 101 5.2.2 Solutions and Chemicals ······································································································ 102 5.2.3 Statistical Analysis ················································································································ 102 5.3 Results ·············································································································································· 102 5.3.1 Vascular Constriction and COX Activity ········································································· 102 5.3.2 Immunohistochemical Staining Analysis of COX1 and COX2 Expression in the Thoracic Aorta and Aortic Root···································································································· 105 5.3.3 Urine 2,3-dinor-6-keto PGF1α Production ········································································ 110 5.3.4 Real-Time RT-PCR of the mRNS expression of COX1, COX2 and PGI2 in the Heart ······························································································································································ 110 5.4 Discussion ······································································································································· 111 CHAPTER 6: EXPOSURE TO DIESEL EXHAUST ENHANCES SOLUBLE GUANYLATE CYCLASE EXPRESSION IN APOE KNOCKOUT MICE ·················· 116 6.1 Introduction ····································································································································· 116 6.2 Methods and Materials ················································································································· 118 6.2.1 Methods ··································································································································· 118 6.2.2 Solutions and Chemicals ······································································································ 119 6.2.3 Statistical Analysis ················································································································ 119 6.3 Results ·············································································································································· 120 6.3.1 Endothelial Function ············································································································· 120 6.3.2 Immunohistochemical Staining of eNOS and sGC Expression in the Thoracic Aorta ······························································································································································ 123 6.3.3 Measurement of Plasma Nitrite and Urine cGMP Concentration ······························· 125 6.3.4 Real-Time RT- PCR of eNOS and sGCα1 mRNA Expression · ·· ··· ·· ·· ·· ··· ·· ·· ·· ··· ·· ·· · 126 6.4 Discussion ······································································································································· 127 CHAPTER 7: SUMMARY AND CONCLUSIONS ······························································ 131 7.1 Relationship between DE Inhalation and Atherogenesis ······················································ 132 7.2 Mechanisms Underlying DE Exposure-induced Atherogenesis ·········································· 133 7.2.1 Increased iNOS Expression ································································································· 133 7.2.2 Increased COX2 Expression································································································ 135 7.2.3 Vascular Endothelial Function ···························································································· 136 7.3 Limitations ······································································································································ 141 7.4 Future Directions ··························································································································· 141 ix  BIBLIOGRAPHY ······························································································································ 142 APPENDIX A: EXPRESSION OF PROTEOGLYCAN IN AORTIC ROTT LESION ·································································································································································· 169 APPENDIX B: EFFECT ON THE THICKNESS OF SMOOTH MUSCLE IN TORAACIC AORTA ························································································································ 170 APPENDIX C: ASSOCIATION BETWEEN INOS EXPRESSION AND CELLULAR FEATURES OF ATHEROSCLEROTIC PLAQUE IN AORTIC ROOT ······················· 171 APPENDIX D: NO CHANGES IN VASOCONSTRICTION IN THE PRESENCE OF TXA2 INHIBITOR ···························································································································· 172 APPENDIX E: VASCULAR FUNCTION ANALYSIS OF COX1 AND COX2 ACTIVITY ··········································································································································· 173 APPENDIX F: NO MODIFICATION OF DE EXPOSURE ON THE EXPRESSION OF SGCβ1 IN THE THORACIC AORTA ·············································································· 174 APPENDIX G: CONCENTRATION-RESPONSE CURVE OF CLONIDINEINDUCED VASORELAXATION ································································································ 175 APPENDIX H: VASCULAR FUNCTION ANALYSIS OF ENDOTHELIN-1 ACTIVITY ··········································································································································· 176 APPENDIX I: CORRELATION BETWEEN INOS EXPRESSION AND BAND CELLS ··················································································································································· 177 APPENDIX J: VASCULAR FUNCTION ANALYSIS AND IMMUNOHISTOCHEMISTRY ANALYSIS OF COX AND INOS ACTIVITY AND EXPRESSION ····································································································································· 178 APPENDIX K: VASCULAR FUNCTION ANALYSIS OF THE EFFECT OF INOS ON ACH RELAXATION ················································································································ 179  x  List of Tables  Table 1.1 Relationships between particle surface area, numbers and size ·································· 3 Table 1.2 Summary of recent multi-city analyses and studies ······················································· 8 Table 1.3 Summary of recent multi-city analyses and studies for the effects of chronic PM exposure and cardiovascular effects ··································································································· 11 Table 2.1 Composition of Diesel Exhaust Particles (DEP) generated at the Northlake exposure facility ······································································································································ 49 Table 3.1 Plasma cholesterol and triglyceride level ······································································· 63  xi  List of Figures Figure 1.1 The size of different fractions of PM10············································································ 2 Figure 1.2 Where does PM10 go in the respiratory system ····························································· 4 Figure 1.3 Comparison of PM10 and PM2.5 Sources ········································································· 5 Figure 1.4 Diagrammatic representations of the hypothetical events after exposure to ultrafine particles (right) compared with fine particles (left) ·························································· 6 Figure 1.5 Risk estimates provided by several cohort studies per increment of 10 µg/m3 in PM2.5 or PM10 ·········································································································································· 12 Figure 1.6 Biological pathways linking PM exposure with cardiovascular diseases ·············· 13 Figure 1.7 Steps in the recruitment of mononuclear phagocytes to the nascent atherosclerotic plaque ························································································································································ 25 Figure 1.8 The timeline for endothelial dysfunction and the development of atheroslcerosis ···································································································································································· 27 Figure 1.9 NO-mediated vasorelaxation ··························································································· 29 Figure 1.10 Peroxynitrite (ONOO) induced apoptotic and necrotic cell death ························ 35 Figure 3.1 Exposure effects ················································································································· 64 Figure 3.2 Circulating band cell counts and their relationship with alveolar macrophages ·· 64 Figure 3.3 Movat staining analysis of atherosclerotic lesion size, cellularity and the number of foam cells in aortic root ···················································································································· 67 Figure 3.4 Histochemical analysis of the components of atherosclerotic plaque in aortic root ···································································································································································· 69  xii  Figure 3.5 Immunohistochemical analysis of the expression of oxidative stress markers in the aortic root lesions ····························································································································· 72 Figure 3.6 Association of DE exposure, plaque foam cell formation and the oxidative stress markers······················································································································································ 73 Figure 3.7 Analysis of systemic oxidative stress ············································································ 74 Figure 3.8 Plasma myeloperoxidase (MPO) levels ········································································ 74 Figure 4.1 Reduced vasoconstriction and increased iNOS activity in the thoracic aorta ······· 87 Figure 4.2 Immunohistochemical analysis of iNOS expression in the thoracic aorta············· 88 Figure 4.3 Immunohistochemical and Western blotting analysis of iNOS expression in the heart ··························································································································································· 90 Figure 4.4 iNOS expression in atherosclerotic plaque in aortic root and its relationship with the expression of CD36 and nitrotyrosine ························································································· 92 Figure 4.5 Increased mRNA expression of iNOS and NF-κB in the heart ······························· 93 Figure 4.6 Increased NF-κB activity of heart nuclear extract ······················································ 94 Figure 5.1 Reduced vasoconstriction in the thoracic aorta ························································· 103 Figure 5.2 Reduced vasoconstriction in the presence of COX inhibitor (indomethacin) and reduced fractional constriction in the thoracic aorta ····································································· 104 Figure 5.3 Representative photomicrographs of staining in the thoracic aorta ······················ 106 Figure. 5.4 Immunohistochemical analysis of COX1 and COX2 expression in the thoracic aorta ························································································································································· 107 Figure 5.5 Representative photomicrographs of staining in aortic root ··································· 108 Figure 5.6 Immunohistochemical analysis of COX1 and COX2 expression in aortic root · 109 xiii  Figure 5.7 Urine 2,3-dinor-6-keto PGF1α concentration ····························································· 110 Figure 5.8 The mRNA expression of COX1, COX2 and PGI2 in the heart ···························· 111 Figure 6.1 Concentration-response curves show that ACh-stimulated endotheliumdependent and SNP-elicited endothelium-independent relaxation············································· 121 Figure 6.2 The effect of NOS inhibitors on PE-induced vasoconstriciton in the thoracic aorta ·································································································································································· 122 Figure 6.3 Immunohistochemical analysis of eNOS and sGCα1 expression in the thoracic aorta ························································································································································· 124 Figure 6.4 Measurement of plasma nitrite concentration and urine cGMP level ··················· 125 Figure 6.5 mRNA expression of eNOS and sGCα1 in the abdominal aorta ·························· 126 Figure 7.1 Summary of the signalling pathways contributing to DE inhalation-induced atherogenesis ········································································································································· 139  xiv  List of Abbreviations and Acronyms ACh ANOVA BH4 Ca2+ [Ca 2+ ]i CaCl2 cGMP COX DE ELISA EC50 ECE EDRF EDTA et al ET-1 ETA ETB FA GM-CSF GTP HPRT1 ICAM-1 IHC iNOS IL-1 IP3 K+ KCl kDa L L-NAME M MgCl2 MI MIP NaCl NF-κB NO NOS NSAIDs NT OxLDL PE PGH2 PKC  acetylcholine analysis of variance terahydrobiopterin calcium ion intracellular Ca 2+ concentration calcium chloride cyclic guanosine 3',5'-monophosphate cyclooxygenase diesel exhaust enzyme-linked immunosorbent assay effective dose eliciting 50 % response endothelin-converting enzymes endothelium-dependent relaxing factor ethylenediaminetetraacetic acid et alii (Latin, ‘and others’) endothelin-1 endothelin type A receptor endothelin type B receptor filtered air granulocyte-macrophage colony-stimulating factor guanosine 5’-triphosphate hypoxanthine phosphoribosyltransferase-1 intercellular adhesion molecule 1 immunohistochemistry inducible nitric oxide synthase interleukin-1 inositol-1,4,5-trisphosphate potassium ion potassium chloride kilodalton litre(s) NG-nitro-L-arginine-methyl ester mole⋅l-1 magnesium chloride myocardial infarction macrophage inflammatory protein sodium chloride nuclear factor-kappa B nitric oxide nitric oxide synthase nonsteroidal anti-inflammatory drugs nitrotyrosine oxidized low-density-lipoprotein phenylephrine prostaglandin H2 protein kinase C xv  PGI2 PM10 PMN PSS ROS RT-PCR SEM sGC SNP SOD TNF TXA2 VCAM-1 VSMC  prostagcyclin I2 particulate matter air pollution with diameter less than 10µm polymorphonuclear leukocyte physiological salt solution reactive oxidative species real-time reverse transcription polymerase chain reaction standard error of the mean soluble guanylate cyclase sodium nitroprusside superoxide dismutases tumour necrosis factor thromboxane A2 vascular adhesion molecule-1 vascular smooth muscle cell  xvi  Acknowledgements I would like to thank my thesis advisors, Drs. Stephan van Eeden and Cornelis van Breemen, for the experience I have gained working with them, their knowledge, guidance, and profound insight have sharpened my critical thinking during the course of my research. I would also like to thank the members of my committee: Drs. Aly Karsan, Darryl Knight, David Granville for reading my thesis and offering advice.  I would like to send my deepest thanks to my family: my parents, my brother and my husband who always stand by me and without whom none of this would have been possible. Their unconditional love and support are the most invaluable thing I have had in my life.  I would like to acknowledge the financial support from Natural Sciences and Engineering Research Council of Canada, Michael Smith Foundation for Health Research, and Heart and Stroke Foundation of Canada.  xvii  Dedication  To my parents, Baosheng Bai and Jinli Wu, my husband, Jie Cai, and my daughter, Annie Yueye Cai  xviii  Chapter 1 Introduction  1.1 Background  An association between ambient particulate matter (PM) and mortality was first revealed in December 1930 in the Meuse Valley air pollution episode, in which 60 people died within 3 days in Engis, a town of 3,500 inhabitants in Belgium (1). Studies following the “London Fog Disaster” in December 1952 provided the first solid epidemiological data showing that exposure to PM increases mortality with an excess of 4,000 deaths over 2 weeks (2). Since then, numerous studies have supported the notion that chronic exposure to PM is an independent risk factor for respiratory as well as cardiovascular morbidity and mortality (3-10). In addition, evidence has shown that cardiovascular events can occur within hours to days after exposure to PM. The levels of ambient PM are positively correlated with mortality and morbidity rates from respiratory and cardiovascular diseases. Moreover, respiratory morbidity and mortality are related to admissions for pneumonia, asthma, pulmonary emboli, and chronic obstructive pulmonary disease (COPD) (11-14), while cardiovascular morbidity and mortality are attributable to acute coronary syndrome, arrhythmia, heart failure, and stroke (3; 7; 8-10). Major improvements in air quality have occurred over the last 50 years, yet the association between ambient PM and cardiovascular mortality is still evident, even when pollution levels are below current national and international targets for air quality. No apparent threshold exists below which the association no longer applies (11). Although the  11  1  risk to a given individual at any single time point seems small, given the prodigious number of people being continuously exposed, particulate matter air pollution imparts a tremendous burden to the global public health, and is ranked as the 13th leading cause of mortality. World Health Organization (WHO) estimates that ambient PM is responsible for three million deaths each year (World Health Organization (2002) World Health Report. Geneva [http:www.who.int/whr/2002/en]).  1.1.1 Particulate Matter Air Pollution (PM10) Air pollutants are a heterogeneous mixture of gases and particulate matter. The major gaseous components of air pollution are NO2, CO, SO2, and ozone. Particulate matter air pollution consists of solid and liquid particles derived from vehicle emissions, forest fires, and industrial, domestic and agricultural pollutants. Particulate matter air pollution with diameter less than 10 µm is collectively known as PM10. Based on the size, PM10 is subdivided into coarse particles (PM2.5-PM10, diameter 2.5-10µm), fine particles (PM0.1PM2.5, diameter 0.1-2.5µm) and ultrafine particles (diameter <0.1µm) (Fig 1.1).  Figure 1.1 The size of different fractions of PM10 2  Inhaled particles are cleared from the lungs via phagocytosis by alveolar macrophages or via translocation to the tracheobronchial lymph nodes. Chronic inhalation of poorly soluble ambient particles can cause progressive accumulation of aggregated, particle-laden macrophages and lead to lung overload, after the particle deposition rate exceeds that of the clearance by alveolar macrophages (12). Particulate matter air pollution larger than 10 µm is generally filtered by the defense system in the nose and throat, with particles less than 10 µm being inhaled deep into the bronchi and lungs. Particles with larger diameters tend to settle more quickly, whereas fine and ultrafine particles (UFPs) remain suspended in the air for a longer period, resulting in greater alveolar deposition efficiency. Compared with coarse particles, fine particles and UFPs deposit in greater numbers and into deeper regions of the lungs due to the larger surface area (Fig 1.2). A collection of all particles of these three different particle diameters (Table 1.1) would always give the same airborne mass concentration of 10 µg/m3. Nevertheless, the physical characteristics of the cloud are very different for the particle number and surface area, and both of these are properties that are believed to have an important impact on the lung (13) .  Table 1.1 Relationships of the particle surface area, numbers, and size. (Adapted with permission from Donaldson et al, Occup Environ Med., 2001) (15). Particle diameter (µm) 2  Particles/ml of air 1.2  Particle surface area (µ3/ml air) 24  10  0.5  153  120  10  0.02  2400000  3016  Airborne mass concentration (µg/m3) 10  3  Particles bigger than PM10 Nose and Throat remove PM10 Trachea & Upper Bronchi deposition  PM2.5 & UFP Bronchioles and Alveoli deposition  Figure 1.2 Where does PM10 go in the respiratory system. (Adapted from the websites of the National Cancer Institute).  Fine particles and UFPs are mainly generated from the combustion of fossil fuels used in transportation, manufacturing, and power generation. Although diesel engines generate up to 25% less CO2 (a green house gas implicated in global warming), they emit up to 100-150 times more particles than do gasoline engines (14). Diesel exhaust (DE) is a major contributor to fine particles, accounting for up to 90% of the fine particulate mass in ambient air of many major cities, such as London (15; 16). In North America cities, the contribution of DE to ambient particulate matter is less than that in European cities (Fig 1.3).  4  Figure 1.3 Comparison of PM10 and PM2.5 Sources in Washington, DC. (The pie chart is based on analysis of actual ambient air samples taken during 1992-95 in Washington, DC.) (Adapted from IMPROVE, Cooperative Center for Research in the Atmosphere CSU, Ft. Collins, CO, July 1996).  Many of these individual components of atmospheric PM10 are not especially toxic at ambient levels and some major constituents, such as sodium chloride, are harmless. In contrast, DE consists of a central carbon core nucleus onto which an estimated 18,000 combustion products are absorbed, including organic chemicals like polycyclic aromatic hydrocarbons, and oxidized transition metals (17; 18). Toxicological studies have shown that exposure to fine and UFPs causes greater inflammatory effects than large ones of the same material. Although the mechanism remains unclear, evidence indicates that the large surface area of these fine particles and the oxidative stress associated with the transition metals carried by UFPs play essential roles in the strong association between exposure to PM10 and health effects (19; 20). In addition, UFPs can evade from being cleared-up or phagocytosed by alveolar macrophages, resulting in more opportunities for UFPs to interact with lung cells, and lead to secondary effect in the lung (Fig 1.4). 5  Figure 1.4 Diagrammatic representations of the hypothetical events after exposure to ultrafine particles (right) compared with fine particles (left). The essential elements of the ultrafine response are many particles outside and inside macrophages. Release of mediators from the macrophages and epithelial cells due to activation of signaling pathways mediated by oxidative stress, may then lead to inflammation. The enhanced interaction of particles with the epithelium leads to their transfer to the interstitium. (Adapted with permission from Donaldson K et al. Occup Environ Med. 2001) (20).  The typical background concentrations of PM10 in North America or Western Europe are 20-50 µg/m3, and these concentrations increase to 100-250 µg/m3 in industrialized areas and in the developing world. The most recent National Ambient Air Quality Standards issued by the U.S EPA in 2006 for PM10 are the annual standard of 55 µg/m3, and the 24hour standard of 150 µg/m3. The standards for PM2.5 levels have been strengthened to 15 µg/m3 (annual) and 35 µg/m3 (24-hour) based on vast evidence showing a strong association between fine particles and cardiovascular diseases.  6  1.1.2 Epidemiological Evidence for Adverse Cardiovascular Effects Associated with Exposure to Particulate Matter Air Pollution (PM10) Evidence from numerous epidemiological studies conducted in different countries across the continents indicates a strong association between increases in PM10 and greater acute (Table 1.2) and chronic cardiovascular morbidity and mortality (Table 1.3).  1.1.2.1 Acute Cardiovascular Effects Induced by PM10 Exposure Burnett and colleagues monitored PM10 levels for 300,000 elderly residents in 21 US cities between 1985-1999, and found that a 10 µg/m3 increase in PM10 was associated with a significantly increased risk of hospitalization for myocardial infarction (MI), which occurred within a few hours after exposure (21). Studies examining daily PM10-related mortality effects were also conducted in multiple cities in Europe and Asia, and the results consistently showed that exposure to PM10 was significantly associated with daily mortality accounts for all-causes and cardiovascular mortality (Table 1.2). The overall evidence from a time-series analysis conducted worldwide confirms the existence of a small, yet consistent association between increased cardiovascular mortality and short-term (1 to 5-day) elevation of PM10 and PM2.5 prior to the cardiovascular events.  7  Table 1.2 Summary of recent multi-city analyses and studies.These studies have been published since 2004 of the comparison of pooled estimated of percent increase (and 95% CI or Posterior Interval or t Value) in RR of mortality estimated across meta-analyses and multicity studies of daily changes in exposure (Modified from Brook RD et al. Circulation. 2010) (22). Studies Percent Increases in Mortality (95% CI)  Meta-estimate with and without adjustment for publication bias (23) Meta-estimates from COMEAP report to the UK Department of Health on CVD and air pollution (24) NMMAPS, 20 to 100 US cities (25) APHEA-2, 15 to 29 European cities (26) US, 6 cities (27)  Exposure (µg/m3) 20 PM10  Increment All-Cause  Cardiovascular  1.0 (0.8–1.2) 1.2 (1.0–1.4)  20 PM10  1.8 (1.4–2.4)  10 PM10 20 PM10  1.4 (0.7–2.2) 0.4 (0.2–0.8)  0.6 (0.3–1.0)1  1.2 (0.8–1.4)  1.5 (0.9–2.1)  10 PM2.5 10 PM2.5  1.2 (0.8–1.6)  1.3 (0.3–2.4)2  1.2 (0.3–2.1)  0.9 (–.1, 2.0)  California, 10 9 cities (29) PM2.5 France 20 9 cities (30) BS Japan, 20 13 cities, age >65 yr SPM (31) Asia, 10 4 cities (32) PM10 US, 10 112 cities (33) PM2.5  0.6 (0.2–1.0)  0.6 (0.0, 1.1)  1.2 (0.5–1.8)3  1.2 (0.2–2.2)3  1.0 (0.8–1.3)  1.1 (0.7–1.5)  0.55 (0.26–0.85)  0.59 (0.22–0.93)  0.98 (0.75–1.22)  0.85 (0.46–1.24)  0.46 (0.21–0.71)  0.32 (0.00–0.64)  US, 27 cities (28)  20 PM10  10 PM10-2.5  CI indicates confidence interval or posterior interval. 1. Cardiovascular and respiratory deaths combined. 2. Ischemic heart disease deaths. 3. Chronic obstructive pulmonary disease deaths. 4. Includes general additive model–based analyses with potentially inadequate convergence. 5. Results for PM10–2.5 are from 47 cities.  8  1.1.2.2 Chronic Cardiovascular Effects Induced by PM10 Exposure Although short-term changes in PM10 concentrations have deleterious health effects, longer-term exposure may have a more pertinent clinical health effect on cardiovascular morbidity and mortality given that individuals are typically exposed to higher air pollution levels over extended periods of time. A 16-year prospective study (Harvard Six City Study), involving 8,000 people living in 6 American cities with different levels of ambient PM (10fold difference between the most and the least polluted cities), showed a strong association between increases in cardiovascular mortality and high PM10 levels after adjusting for cigarette smoking and other major cardiovascular risk factors (34). Another landmark cohort-based mortality study, that enrolled approximately 1.2 million American adults in 3  1982, found that an elevation of 10 µg/m PM2.5 was associated with ~6% increase in cardiovascular mortality (35). These cohort studies (summarized Table 1.3) demonstrate larger overall mortality effects, compared to those of acute exposures (Table 1.2). In addition, exposure to elevated PM levels over the long-term can reduce life expectancy. A recent large study found that a decrease of 10 µg/m3 in the long-term PM2.5 concentration was related to an increase in mean life expectancy around one year (36). That exposure to PM induces cumulative changes causatively linked with mortality is further supported by studies showing that reduction in ambient PM leads to short-term (weeks to months) and long-term (years) decreases in mortality. For instance, a 15 µg/m3 decrease in PM10 concentration in Utah valley (during a 13-month strike at a local steel mill) was associated with a 3.2% decrease in total mortality (6). Similarly, a 35 µg/m3 decrease in mean black smoke concentration attributable to a ban on coal sale in the city of Dublin was accompanied by a 5.7% decrease in non-trauma deaths in 72 months after the ban, compared with the same period before the ban (37). A current update of the Harvard Six City Study extended the mortality follow-up for an additional 8 years showed that a 1 µg/m3 reduction 9  in PM2.5 was associated with a 3% decrease in overall mortality (35; 38). These studies strongly support a causal relationship of exposure to PM and cardiovascular morbidity and mortality.  10  Table 1.3 Summary of recent multi-city analyses and studies for the effects of chronic PM exposure and cardiovascular effects (Modified from Brook RD et al. Circulation. 2010) (22). Percent Increases in Mortality (95% CI) Studies Size of Follow- Covariates All-Cause Cardiovascular Cohort Up Controlled for (000s) Period Harvard Six ~8 1974– Individual 13 Cities, 1991 (smoking+others) (4.2–23) original (3) Harvard Six- ~8 1974– Individual 16 28 Cities, 1998 (smoking+others) (7–26) (13–44) extended (38) Six-Cities ~340 2000– Individual 21 Medicare 2002 (age, sex) (15–27) cohort (39) ACS, ~500 1982– Individual 6.6 Original (7) 1989 (smoking+others) (3.5–9.8) ACS, HEI ~500 1982– Individual 6.2 12 extended 1998 (smoking+others) (1.6–11) (8–15) (40) +ecological US Medicare ~132000 2000– Individual 6.8 cohort (41) 2005 (age, sex) (4.9–8.7) 1 +ecological +COPD Women’s ~66 1994– 76 (25–147), Health 2002 24 (9–41)2 Initiative (42) French ~14 1974– Individual 7 PAARC (43) 2000 (smoking+others) (3–10)2 German ~5 1980s, Individual 12 women (44) smoking and (–8 to 38) 1990s– socioeconomic 2003 status Oslo, ~144 1992– Individual age, 10 (5–16),3 Norway (45) 1998 occupational 14 (6–21), class, education 5 (1–8), 3 (0–5) Dutch cohort ~121 1987– Individual 6 4 (46) 1996 (smoking+others) (–3 to 16) (–10 to 21) +ecological Great Britain ~660 1966– Socioeconomic 1.3 1.2 (47) 1998 status (1.0–1.6)4 (0.7–1.7)4 HEI indicates Health Effects Institute; VA, Veterans Affairs; COPD, chronic obstructive pulmonary disease. 1. Three estimates are for the East, Central, and West regions of the United States, respectively. 2. Any cardiovascular event. 3. Four estimates are for men 51–70 y old, women 51–70 y old, men 71–90 y old, and women 71–90 y old, respectively. 4. Associated with 10 µg/m3 British Smoke (BS) or PM10  11  Figure 1.5 Risk estimates provided by several cohort studies per increment of 10 µg/m3 in PM2.5 or PM10. CPD, cardiopulmonary; CVD, cardiovascular disease; IHD, ischemic heart disease. (Adapted with permission from Brook RD et al. Circulation. 2010) (22).  Collectively, the evidence from studies conducted independently in different countries at different periods of time indicates that an elevation of PM10 levels is strongly associated with increases in cardiovascular mortality, attributable to atherosclerosis, dysrhythmias, congestive heart failure, ischemic heart disease, and stroke (9; 48-56) (Fig.1.5). Of all common air pollutants, fine ambient particles have the strongest association with mortality (3; 57-59). In addition, having pre-existing cardiovascular risk factors such as coronary heart diseases, heart failure, diabetes mellitus and ageing increases the probability of cardiovascular mortality following PM10 exposure (60; 61).  12  Figure 1.6 Biological pathways linking PM exposure with cardiovascular diseases. The 3 generalized intermediary pathways and the subsequent specific biological responses that could be capable of instigating cardiovascular events are shown. MPO indicates myeloperoxidase; PAI, plasminogen activator inhibitor; PSNS, parasympathetic nervous system; SNS, sympathetic nervous system; and WBCs, white blood cells. A question mark (?) indicates a pathway/mechanism with weak or mixed evidence or a mechanism of likely yet primarily theoretical existence based on the literature. (Adapted with permission from Brook RD et al. Circulation. 2010) (22).  1.1.3 Proposed Pathways for PM10–induced Cardiovascular Effects The biological pathways by which exposure to PM10 exerts cardiovascular effects have been a major focus of research over the past decade. Although the precise pathways are still not fully understood, two hypotheses have been proposed based on population and animal studies. Seaton and colleagues (1995) proposed that the deposition of ambient PM in the lung provoks a low-grade alveolar inflammation with a secondary systemic inflammatory response resulting in downstream cardiovascular death, especially in susceptible individuals 13  (62). Since then, numerous authors have supported this hypothesis and further expanded it, reporting that exposure to PM10 causes changes in plasma viscosity (63), and an increase in the procoagulant state, including elevated levels of fibrinogen (64; 65), platelet activation (66), a pro-inflammatory state of blood (67) that includes bone marrow stimulation with an increase in circulating white cells (68), polymorphonuclear neutrophils (PMNs), and monocytes (69; 70). In addition, to explain the relative short interval between increases in PM10 exposure and cardiovascular events, Godleski et al. suggested that fine particles or UFPs could enter the blood and adversely affect the heart by initiating arrhythmias and sudden death in susceptible subjects (71). Since then, several studies have demonstrated that UFPs have the ability to translocate into the circulation both in animal models and in humans (71-74). These studies suggest that a direct effect of translocated UFPs on the vasculature could result in observed increases in heart rate variability, increases in heart rate, changes in blood pressure (75), and enhanced susceptibility to arrhythmias and modified autonomic or other neurological reflexive effects (76) associated with exposure to PM10. These two hypotheses are not mutually exclusive, and evidence from both human and animal studies indicates that the biological response to inhaled ambient particles is very complex; hence both mechanisms may contribute to the adverse cardiovascular effects of PM10 exposure (Fig.1.6). 1.1.3.1 PM10-Induced Pulmonary and Systemic Inflammation Substantial evidence indicates that inhaled particles provoke an inflammatory response in the lungs with a consequent release of prothrombotic and inflammatory cytokines into the circulation that leads to exacerbations in cardiovascular disease (22; 77).  14  1.1.3.1.1 Lung Inflammatory Response Induced by PM10 Alveolar macrophages and epithelial cells of the bronchial wall, and alveolar spaces are exposed to inhaled particles, and are the principle cell types that process inhaled ambient particles in the lung. In processing these particles, the cells produce pro-inflammatory mediators that are capable of eliciting both local and systemic inflammatory responses (78; 79).  1.1.3.1.1.1 Alveolar Macrophage Response to PM10 Exposure As a function of self and immune-defense, upon deposition of particles in the lung, alveolar macrophages produce increasing amounts of pro-inflammatory mediators; in particular, interleukin (IL)-1β, IL-6, IL-8, macrophage inflammatory protein (MIP)-1α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (79-82). IL-1β is one of the “acute response” cytokines released by alveolar macrophages and epithelial cells. IL-1β has a broad stimulating effect on B cells and T cells, and helps to propagate the extension of a local or systemic inflammatory process. IL-6 contributes to the initiation and extension of the inflammatory process, promoting monocyte differentiation toward macrophages (83), activation of B and T lymphocytes, and stimulates hepatocytes to produce acute phase proteins (84). In addition, IL-6 increases granulocyte release into the circulation from the bone marrow, and promotes granulocyte sequestration in microvascular beds (85). IL-6 also increases the production of reactive oxygen species (ROS), and regulating cell adhesion properties by the activation of peripheral blood mononuclear cells to secrete monocyte chemoattractant protein (MCP)-1 (86). IL-8 is a critically important chemoattractant and activator facilitating the recruitment of both polymorphonuclear leukocytes (PMN) and monocytes into the airspace. MIP can activate granulocytes, and can lead to acute neutrophilic inflammation. MIP also induces the synthesis and release of other pro15  inflammatory cytokines, including IL-1 and IL-6. GM-CSF is not only a hematopoietic growth factor but also has an important granulocyte degranulation factor that enhances tissue damage induced by granulocytes (87). IL-10, a cytokine known to inhibit the production of pro-inflammatory factors such as TNF-α, IL-1β, IL-6, and IL-8, is not stimulated by particle exposure (79), suggesting that PM10 does not directly induce a significant anti-inflammatory cytokine response. Collectively, the evidence suggests that in response to the exposure of ambient particles, alveolar macrophages release a collection of inflammatory mediators that can elicit a local inflammatory response in the lung, and promote leukocyte recruitment into lung tissues and air space, leading to lung inflammation. Exposure to ambient particles also modulates the responses of alveolar macrophages to infectious agents. Exposure of human alveolar macrophages to diesel exhaust particles (DEP) significantly suppresses the alveolar macrophage responsiveness to gram-negative (lipopolysaccharide, LPS) and positive (lipoteichoic acid) bacterial products. The impaired alveolar macrophage responsiveness may also contribute to compromised pulmonary defense mechanisms, further explaning the frequently reported association of increased lung infections with exposure to episodes of increased air pollution (88). The suppressed alveolar macrophage responsiveness can be attenuated by scavengers of free radicals, indicating that this is a free radical-mediated process (89). In addition, the compromised alveolar macrophage action is specific in response to DEP exposure and has not been observed with other ambient particles such as carbon black or EHC93 (79; 91), suggesting that the composition of particles plays an important role in the reaction of lung cells (e.g, alveolar macrophages) to inhaled particles. In summary, alveolar macrophages are crucially important in initiating and promoting the lung inflammatory response after exposure to ambient PM.  16  1.1.3.1.1.2 Lung Epithelial Cell Response to PM10 Exposure The surface area of the lungs is equivalent to the size of a tennis court, and is covered with epithelial cells. Because of its large surface area, lung epithelial cells bear the burden of exposure to inhaled particles and are essential in processing ambient particles. Several studies have shown that exposure of different cell lines of lung epithelial cells to PM10 can produce various pro-inflammatory mediators (91-94). Studies using primary cultures of human bronchial epithelial cells (HBEC) revealed that these cells produced IL-1β, IL-8, GM-CSF, and leukemia inhibitory factor (LIF) in a dose-dependent manner in both mRNA and protein levels (91). The cytokine production in the human epithelial cells was attenuated by chelating transitional metals from urban particles, suggesting that metal cations augment the cellular responses of ambient particles (95; 96). In comparison with bronchial epithelial cells, production of cytokines, such as IL-1β, IL-6, GM-CSF, and LIF by particle exposure to HBEC, is absent in A549 cells, a cell line representing alveolar epithelial cells. The A549 cells tend to produce IL-8 and macrophage chemoattractant protein (MCP)-1, which are not only important chemoattractants of PMN and monocytes, but are also able to up-regulate the expression of intercellular adhesion molecule-1 (ICAM-1) on their surface, thus contributing to leukocyte recruitment into the airspace. These studies suggest that bronchial and alveolar epithelial cells respond differently to ambient particles.  1.1.3.1.1.3 Alveolar Macrophage and Lung Epithelial Cell Interaction  The close proximity of alveolar macrophages and epithelial cells in the lung increases the likelihood of the two cell types interacting and amplifying their responses to PM10 exposure. Mediators such as IL-1β and TNFα released from alveolar macrophages upon exposure to PM10 can, in turn, activate epithelial cells, causing them to enhance their mediator production and release. Supernatants of alveolar macrophages exposed to PM10 17  increase the expression of IL-1β, LIF, and IL-8 mRNA in HBEC to a greater extent than when do supernatants from alveolar macrophages exposed to control medium (81). Cocultures of human alveolar macrophages and bronchial epithelial cells synergistically produced GM-CSF and IL-6, both of which are involved in the systemic inflammatory response to inhaled PM (92). The interaction of alveolar macrophages with lung epithelial cells promotes the production of mediators responsible for the release of monocytes from the bone marrow, an important effector cell involved in lung inflammation and the progression of atherosclerosis associated with chronic exposure to PM (97). Recent findings suggest that epithelial cells can also signal other lung cell types. For example, mediators from HBEC exposed to DEP induce maturation of dendritic cells, possibly via the up-regulation of GMCSF (98). Therefore, PM10-induced interactions of lung cells could be signaled via direct adhesion contact between these cells or via mediators functioning in a paracrine fashion. Collectively, these studies demonstrate that lung cells phagocytose inhaled ambient particles and produce mediators that induce and further amplify the local inflammatory responses in the lung. Several of the mediators produced in the lung are implicated in the systemic inflammatory response associated with exposure to PM. In addition, chronic exposure to ambient PM could establish a dynamic biological association between particles and chemokine production, providing a source for sustained inflammatory responses, which can constantly recruit inflammatory cells, such as monocytes and neutrophils into the circulation and to interact with the blood vessel wall (99-101). It is worthy noting that the concentrations of PM used in some of the studies(100; 102) were very high and not environmently relavent, therefore, the mechanisms involved may be not the same as what lower-level ambient inhalation exposures(101).  18  1.1.3.1.2 Systemic Inflammatory Response Induced by PM10 Exposure The systemic inflammatory response induced by exposure to ambient particles occurs in three phases: 1) release of inflammatory mediators into the circulation; 2) activation of the acute phase response; and 3) stimulation of bone marrow with mobilization and activation of inflammatory cells.  1.1.3.1.2.1 Circulating Mediator Release Induced by PM10 Exposure An increase in circulating cytokines and/ or proinflammatory mediators, activated by immune cells, could serve as a mechanism to instigate adverse effects on the heart and vasculature. Numerous experiments have demonstrated increased cellular and inflammatory cytokine content, for example, IL-6, IL-1β, TNFα, interferon-γ, and IL-8, of bronchial fluid, and sometimes, in circulating blood after acute exposure to a variety of pollutants (79; 81; 102-106). Exposure of healthy young volunteers to an acute elevation of PM10 induced increases of blood cytokine levels that promptly declined when the pollutants were cleared (68; 79) albeit that other cuases, including stress, increased physical activity, weather and temperature changes, might not be fully excluded. The positive correlation between reduced circulating cytokine levels and decreased PM10 levels supports a strong link between exposure to PM and the systemic inflammatory response. The cytokines (GM-CSF, IL-6, and IL-1β) that increased in the circulation were similar to those produced by alveolar macrophages exposed to urban particles ex vivo, suggesting a link between lung inflammation and the systemic response elicited by air pollution. In support of this finding, Tamagawa and colleagues found that in vivo exposure of rabbits to ambient particles (ECH93) led to increases in circulating levels of IL-6, which was positively correlated with the number of activated alveolar macrophages and the total alveolar macrophages that 19  phagocytosed particles (104). This suggests an important role of alveolar macrophages in instigating the systemic inflammatory response. We recently demonstrated that IL-6 produced in the lung after PM10 exposure translocated to the circulation and was responsible for inducing systemic inflammation and vascular endothelial dysfunction (107).  1.1.3.1.2.2 Acute Phase Response Induced by PM10 Exposure Part of the systemic response to exposure to air pollution is the activation of the acute phase response as indicated by an increase in circulating acute phase proteins (108-110). Exposure to ambient PM up-regulates the production of circulating levels of acute phase proteins such as C reactive protein (CRP), fibrinogen, complement, transferrin, haptoglobin, α1-antitrypsin, and ceruloplasmin, which is positively correlated with cytokine production, particularly IL-1β, IL-6, and TNFα (108; 110-111). Whether these acute phase proteins are just markers of vascular disease or they actually play a direct role in the pathogenesis of vascular disease associated with air pollution is unresolved and needs further investigation.  1.1.3.1.2.3 Bone Marrow Stimulation Induced by PM10 Exposure An integral component of the systemic inflammatory response is the stimulation of the hematopoietic system, specifically in the bone marrow, which results in an increase in circulating leukocytes. We observed leukocytosis, an independent predictor of MI (112), in both human (9) and animal (69; 103; 113) studies, in response to PM10 exposure. This leukocytosis was accompanied by stimulation of the bone marrow, as indicated by the increased release of both PMNs and monocytes. Cytokines, released upon PM10 deposition in the lung, also stimulate the bone marrow, leading to a decreased transit time of both monocytes and PMNs through the bone marrow, and accelerated release of these cells into the circulation (69; 92; 97; 113-117). Additional studies showed that monocytes were 20  released from bone marrow at baseline, earlier and more rapidly than were neutrophils (118). The release of monocytes from the marrow was accelerated further by inflammation in the lung induced by atmospheric particles (69). Newly released, bone marrow-derived PMN expresses higher levels of adhesion molecules (119) and contains more damaging granular enzymes that can amplify tissue inflammation. Evidence has shown that leukocytosis is associated with acute coronary syndrome (112). The cytokines that increase in circulation following PM10 exposure are important for inducing the systemic inflammatory response. Both TNF-α and IL-1β are “acute response” cytokines that can induce secondary cytokine production; act as co-stimulating factors in hematopoiesis; activate the vascular endothelium; and induce the acute phase response (84). IL-6 can induce endothelial dysfunction (104; 107), stimulate the liver to produce acute phase proteins (such as CRP and fibrinogen), and enhance the production of platelets (83; 84). In addition, IL-6 contributes to atherosclerosis and MI by activation of macrophages to secrete MCP-1 (120), which is pivotal for monocyte recruitment into tissues and a central mediator of inflammatory events in atherosclerosis (121; 122). The acute phase protein CRP is thought to be directly involved in the inflammatory response and is associated with a significant risk of acute coronary events (123). CRP is able to increase monocyte infiltration into the arterial wall, amplify inflammatory and pro-coagulant responses, and lead to atherogenesis (124-128). In addition, CRP can cause plaque destabilization and rupture by up-regulation of adhesion molecules, such as VCAM-1, ICAM-1, and E-selectin (126; 129; 130). Fibrinogen contributes to thrombus formation during acute coronary syndromes and is also recognized as a long-term risk for MI (131). GM-CSF, a hematopoietic growth factor not only stimulates granulocyte differentiation and release from the bone marrow, but also activates circulating leukocytes and prolongs their survival in the circulation and at inflammatory sites (83). These cytokines up-regulate the secretion of MCP-1 and promote 21  the accumulation of monocytes and T lymphocytes in atherosclerotic lesions, resulting in the progression of atherosclerosis (79; 132; 133). Collectively, pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and GM-CSF) are key molecules that initiate and promote local and systemic inflammatory response, and contribute to the cardiovascular effects induced by PM10 exposure. 1.1.3.2 Extra-Pulmonary Translocation of PM10 The possibility of extra-pulmonary translocation of ambient particles first received direct support from the observation that radioactively labeled UFPs were detected in the blood stream as soon as one minute after exposure and importantly, remained at peak levels for periods of up to 60 minutes (72). In a study, by Nemmar and colleagues, 99mTechnetium-labeled carbon particles were detected in blood after one minute inhalation, suggesting that UFPs may be able to translocate from the lungs directly into the circulation, thus influencing cardiovascular endpoints (73). This notion was supported by several independent studies proposing a number of intriguing putative passages by which inhaled ambient particles travel from the lung into the circulation and gain access to other organs. The proposed loci for this pulmonary extravasation include the fenestrae of sinusoidal endothelial cells (134), the lung-blood barrier (135; 136), and the alveolar-capillary barrier (137). Circulating UFPs can mediate increased synthesis and/or release of fibrinogen via direct contact with hepatocytes (134). UFP translocation provides a plausible explanation for the relatively short interval between exposure to ambient particles and acute coronary events. In addition, UFP translocation could also explain the observation that ambient particles cause adverse effects in critical organs independent of a systemic inflammatory reaction (108; 134; 138). On the other hand, Mills et al reported inhaled Technegas carbon nanoparticles pass directly from the lungs into the systemic circulation in human (139). The discrepancy 22  may be due to the techniques of labelling these particles, and different physical properties of particles. However, whether translocated ambient particulate matter can cause cardiovascular effects are still controversy.  1.1.4 PM10 and Atherosclerosis Blood vessel inflammation plays an important role in the development of atherosclerosis (140), which is the underlying cause of approximately 80% of cardiovascular diseases. Studies have shown that exposure to PM10 causes development and progression of atherosclerosis. Using Watanabe Hereditarily Hyperlipidemic (WHHL) rabbits, an animal model of naturally developing atherosclerosis, Suwa and colleagues demonstrated that a four-week exposure to urban ambient particles (EHC93) caused progression of atherosclerosis in both the aorta and coronary arteries, which was correlated with the number of alveolar macrophages that contained ambient particulate matter, thus foreshadowing a link between ambient particles and atherogenesis (141). This finding is supported by other animal 3  3  (142; 143), and human studies showing that an elevation of 10 µg/m or 20 µg/m in PM2.5 was associated with 5.9% and 12.1% increases in the development of atherosclerosis, respectively (143; 144). A cohort study of 4,494 individuals demonstrated that living in close proximity to a major urban road associate with a 7% higher of coronary artery calcium score, a marker of coronary atherosclerosis (145).  1.1.4.1 Atherosclerosis  1.1.4.1.1 Pathogenesis of Atherosclerosis  Atherosclerosis has been recognized as a chronic inflammatory and multisystemic disorder involving the vascular, metabolic, and immune systems (141). The development of atherosclerosis starts at young age and its prevalence increases with age. Fatty streak 23  formation has been observed in human fetal aorta and is greatly enhanced by hypercholesterolemia. Intimal accumulation of low-density lipoprotein (LDL) and oxidized LDL (oxLDL) causes endothelial dysfunction, resulting in impairment in vasorelaxation, increases in oxidative stress, and up-regulation of adhesion molecules. These adhesion molecules, including intercellular adhesion molecule (ICAM)-1, vascular adhesion molecule-1 (VCAM-1), and selectins promote leukocytes to interact, adhere to the endothelium, and transmigrate into the tunica intima to become macrophages. Macrophages phagocytose lipid deposited in the intima through several receptors, such as the scavenger receptor CD36. Macrophages take up modified LDL through CD36 and become foam cells. The formation of foam cells is the hallmark of atherosclerosis. Repeated cycles of inflammation promote the accumulation of macrophages/foam cells that leads to the formation of fatty streak and produces proinflammatory cytokines as well as a variety of mediators. The cytokines and mediators, including growth factors, can activate neighboring smooth muscle cells to replicate and migrate, contributing to lesion growth and the synthesis of components of the extracellular matrix, leading to plaque progression towards advanced lesions (146) (Fig 1.7). The advanced lesions are mainly composed of lipids, inflammatory cells, (such as macrophages), smooth muscle cells, and less extracellular matrix (collagen and proteoglycans) (147). In addition, the macrophages and smooth muscle cells release metalloproteinases, which are proteolytic enzymes that can break down collagen in a fibrous cap and leave the plaque prone to rupture, which results in thrombosis. Recent evidence indicates that plaque composition, rather than plaque size, is the real determinant of the plaque’s evolution toward rupture (148). Not surprisingly, a vulnerable plaque has been associated with a high incidence of cardiovascular events, such as MI and stroke (149; 150).  24  Figure 1.7 Steps in the recruitment of mononuclear phagocytes to the nascent atherosclerotic plaque. The steps are depicted in an approximate time sequence proceeding from left to right. The normal arterial endothelium resists prolonged contact with leukocytes including the blood monocyte. When endothelial cells undergo inflammatory activation, they increase their expression of various leukocyte adhesion molecules. Eventually the macrophages congregate in a central core in the typical atherosclerotic plaque. Macrophages can die in this location, some by apoptosis, hence producing the so-called 'necrotic core' of the atherosclerotic lesion. VCAM-1: Vascular cell adhesion molecule-1; MCP-1: monocyte chemoattractant protein-1; CCR2: MCP-1 receptor; MMPs: matrix metalloproteinases. (Adapted with permission from Libby P, Nature, 2002.) (204).  1.1.4.1.2 Apolipoprotein E Knockout Mouse-a Mouse Model for Atherosclerosis  Apolipoprotein E (ApoE) is 1 of 10 apolipoproteins that coat the surface of lipoprotein and mediate the metabolism of lipoprotein particles. ApoE is synthesized primarily in the liver, and plays a crucial role in receptor-mediated removal of lipoproteins from plasma (152). Via the LDL receptor and LDL receptor-related protein, apoE mediates the clearance of very low-density lipoproteins, chylomicrons and their remnants (153). Subjects with apoE 25  deficiency may develop type III hyperlipidemia. In addition to its role in lipoprotein metabolism, apoE has other functions, including antioxidant (154), anti-platelet aggregation (155), anti-proliferation (156), and prevention of the onset of Alzheimer’s disease (157). The apoE gene was the first lipoprotein transport gene to be deleted in mice, and in 1992, the ApoE knockout mice were developed (158). Of the genetically engineered models, the apoE -deficient model is the only one that develops extensive atherosclerotic lesions on a normal chow diet. It is also the model in which the lesions have been characterized most thoroughly. Numerous studies have demonstrated an accelerated atherogenesis in ApoE knockout mice (159). ApoE knockout mice have spontaneous elevations of total plasma cholesterol and triglycerides and reduced HDL. ApoE mice fed with regular chow develop arterial lesions in a time-dependent manner. Lesional distribution is centered in the aortic sinus in young mice, and the lesions are widely distributed throughout the arterial tree by the age of 8-9 months. In young mice (3-4 months), subendothelial foam cell deposits are present in the aortic sinus adjacent to valve attachment sites. By 5 months of age, foam cell deposits, free cholesterol, and mixed smooth muscle cells are present in developing atherosclerotic lesions. After 8-9 months of age, the arterial lesions show increased complexity with the presence of foam cells, smooth muscle cells, and a fibrous cap (160). Atherosclerotic lesions and their development in ApoE knockout mice bear a striking similarity to the process of atherogenesis in humans (161), and hyperlipidemic rabbits (including fat-fed rabbits and Watanabe heritable hyperlipidemic rabbits) (162). Inflammation induces up-regulation of adhesion molecules, the interactions between leukocytes, and the endothelium, migration of leukocytes into the subendothelial space, and conversion of monocytes into macrophages and, ultimately, to foamy macrophages or foam cells (163; 164). The similarities with human lesion formation suggest that this particular mouse model could provide a fertile source for further investigations of atherogenesis. 26  Interestingly while the lesions of ApoE mice develop into fibrous plaques; no evidence suggest that plaque rupture occurs in this mouse model. Another well-established mouse model for atherosclerosis is the LDL receptordeficient model, which has elevated LDL levels and develops advanced lesions on the Western-type diet. Nevertheless, LDL receptor-deficient mice have no lesions, or have only very small lesions, if they are fed a chow diet.  1.1.5 Mediators that Contribute to the Development of Atherosclerosis Induced by Exposure to PM10 1.1.5.1 Endothelium and Nitric Oxide Regulation, and Cardiovascular Dysfunction  Figure 1.8 The timeline for endothelial dysfunction and the development of Atheroslcerosis (Adapted with permission from Stary HC, et al.Circulation,1995 (165).  27  The vascular endothelium is a single layer of cells representing the interface that separates the circulating blood from the rest of the vascular wall. The endothelium is a dynamic structure of cells that secrete multiple mediators (such as nitric oxide, NO) to control vascular tone, maintain vascular integrity, modulate platelet activity, influence thrombogenicity, and prevent cell migration and proliferation. The most studied aspect of endothelial function is the regulation of vascular tone. Endothelial cells contribute to the regulation of vascular tone by releasing several vasoactive factors, including potent vasodilators (such as NO, endothelium-derived hyperpolarizing factor, and prostacyclin), and vasoconstrictors (such as endothelin (ET)-1, thromboxane A2 (TXA2), prostaglandin H2,), radical superoxide anion (O2-), and components of the renin-angiotensin systems). NO was discovered by Furchgott and Zawadzki as a "endothelium-derived relaxing factor" (EDRF) in 1980 (166). NO is synthesized by a family of enzymes called NO synthase (NOS). The human genes for the NOS isoforms are officially categorized in the order of their isolation and characterization; thus, human genes encoding neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) are also termed NOS1, NOS2, and NOS3, respectively. eNOS is constitutively expressed by endothelial cells and regulated by transient increases of intracellular Ca2+ concentrations ([Ca2+]i) (167).  28  Agonist (ACh)  Ca2+--Calmodulin  Endothelial cell BH4 L-Arg  eNOS  L-NAME  NO  NO  SNP  Constriction  Vascular smooth muscle cell sGC GTP cGMP  Relaxation  Figure 1.9 NO-mediated vasorelaxation  In a healthy vessel, the endothelium serves as the main source of NO production through eNOS activity. NO relaxes vascular tissue by stimulating soluble guanylate cyclase (sGC) to increase cyclic guanosine 3',5'-monophosphate (cGMP) levels in smooth muscle cells (221) (Fig 1.9). sGC consists of a larger α (sGCα) and a smaller β (sGCβ) subunit. So far, four sGC genes have been indentified, encoding the sGC subunits α1, α2, β1 and β2. In many tissues; however, only α1 and β1 (sGCα) subunits appear to be present (169). The N-terminal domains of both the α1 and β1 subunits are essential for the stimulation of the enzyme by NO (170). NO is believed to freely pass through the lipid bilayer into the cytosol, bind to the smaller β1 subunit, and, in turn, activate sGC.  29  In addition to modulating vascular tone, NO influences vascular homeostasis in many ways, including inhibition of smooth muscle cell proliferation, platelet aggregation, platelet and monocyte adhesion to the endothelium, LDL oxidation, expression of adhesion molecules, and endothelin production. NO can also stimulate endothelial cell proliferation and protect endothelial cells from apoptosis. “Endothelial dysfunction” is now synonymous with decreased NO bioavailability, which can result from insufficient supply of co-factor (tetrahydrobiopterin, BH4) and/or substrate (L-arginine) (171), with increased oxidative stress and suppressed antioxidant enzyme activity. BH4 is a key cofactor for all NOS enzymes. In the absence of BH4, NOS produces superoxide, instead of NO (172). Inflammatory cytokines, such as IL-1β or TNF-α, can cause endothelial dysfunction or activation. Activated endothelial cells produce MCP-1, monocyte colony-stimulating factor (M-CSF), and IL-6, which further amplify the inflammatory cascade (173). The activation of the endothelium also attenuates eNOS activity and reduces in NO bioavailability. Reduced NO bioavailability up-regulates VCAM-1 and MCP-1 expression and promotes interactions of endothelial cells with monocytes, causing increased adhesion of monocyte and platelets, and a predisposition of the vessel to develop atherosclerosis (173;174). eNOS knockout mice have been found to be prone to atherosclerosis (175). Endothelial dysfunction can occur prior to the appearance of symptoms of cardiovascular diseases (140), and people with endothelial dysfunction have an increased risk for cardiovascular events, including atherosclerosis, coronary artery disease, diabetes, hypertension, and stroke. PM10-induced endothelial dysfunction has been observed in animal studies showing that an exposure to ambient particles impairs both stimulated and endogenous NO production (176-178). DEP significantly retarded the growth of human pulmonary artery endothelial cells (179). Inflammatory cytokines and increased levels of CRP following 30  exposure to ambient particles can also cause endothelial dysfunction by attenuating NO reactivity and up-regulating the expression of ICAM-1, VCAM-1 and E-selectin (181). Excessive ROS production following exposure to PM10 can rapidly scavenge NO and result in reduced NO bioavailability and endothelial dysfunction (182; 183). As a consequence of increased ROS production following exposure to PM10, lipid oxidation and increased oxLDL can decrease eNOS expression (184). Peroxynitrite, produced by the reaction of superoxide anion with NO can also be a culprit for endothelial dysfunction and induce endothelial cell apoptosis (185-189). In addition, peroxynitrite can be rapidly decomposed to hydroxyl radical and nitrogen dioxide which may lead to cause endothelial injury (190). ET-1 is a potent vasoconstrictor synthesized by the vascular endothelium (191). ET-1mediated vasoconstriction occurs primarily through the ETA receptor located on vascular smooth muscle cells. The ETB receptor located on the vascular endothelium stimulates the production of NO and prostacyclin, and causes vasorelaxation, thus opposing the ETAmediated effects. Elevated plasma levels of ET-1 is another footprint of endothelial dysfunction. A variety of factors can directly stimulate endothelial cells to express ET-1 mRNA and release ET-1. These factors include cytokines, growth factors, and vasoactive substances, such as angiotensin II and vasopressin. ET-1 not only elicits significant vasoconstriction, but also activates monocytes and influences the inflammatory response by stimulating the production of monocyte chemoattractant proteins and modulating leukocyteendothelial cell interactions, which promote the recruitment of leukocytes to the vessel wall (192). Under normal conditions, NO can balance and regulate ET-1 production (193). Nevertheless, plasma ET-1 concentrations are increased in patients with atherosclerosis and coronary artery disease, and are correlated with the severity of many cardiovascular diseases (194; 195). Animals that are exposed to urban PM have up to a 100% elevated levels of endothelin at 2 hours after exposure, and the effect lasts as long as two days (196). This time 31  course of elevated endothelin correlates well with the increase in acute coronary events associated with elevations in atmospheric PM (197). Short-term exposure to concentrated ambient particles caused significantly increased vasoconstriction of human brachial arteries and rat pulmonary resistance arteries due to increases in ET-1 (198; 199). Extended investigation found that particle inhalation potentiated the mRNA expression of preproET-1 and endothelin-converting enzyme (200). Up-regulation of inflammatory cells and cytokines in response to PM10 may stimulate ET-1 synthesis in vascular tissue by down-regulation of angiotensin-converting enzyme in endothelial cells (201; 202). It has been well established that the status of endothelial cell function represents an integrated index of both the overall cardiovascular risk burden and the sum of all vasculature protective factors (203). Interestingly, endothelial dysfunction can be demonstrated in patients with risk factors for atherosclerosis in the absence of atherosclerosis itself (204; 205). These observations lend credence to the concept that endothelial dysfunction is integral to the development and progression of disease. Endothelial dysfunction could significantly contribute to the development of atherosclerosis, acute coronary syndromes, and MI associated with exposure to ambient particles.  1.1.5.2 iNOS and Cardiovascular Dysfunction  iNOS is not detectable in most cells under normal physiological conditions, but is rapidly up-regulated in response to cytokines and/or microbial products. Unlike Ca2+dependent activation of eNOS, the regulation of iNOS is independent of [Ca2+]i (206). The induction of iNOS can be initiated by inflammatory cytokines, such as IFN-γ, TNF-α, or IL1 (207), to trigger a chain of protein phosphorylation and eventually lead to the activation of transcription factor NF-κB, which is responsible for iNOS up-regulation.  32  NF-κB is persistently activated in advanced atherosclerotic lesions and its activation is linked to a wide variety of processes, including inflammation, proliferation, differentiation, and apoptosis (208). Evidence from the analysis of human, mouse, and rat iNOS promoters reveals that NF-κB activation plays a key role in regulating iNOS activity and expression. A classical signaling pathway for NF-κB activation is mediated via phosphorylation of two NF-κB inhibitory proteins, IκBα and IκBβ (209). These proteins bind NF-κB in the cytosol under basal conditions, but are degraded upon phosphorylation; hence releasing active NFκB dimers, which translocate from the cytosol to the nucleus to activate transcription of iNOS and other genes (210). The inhibitory proteins are under complex temporal regulation, allowing for a transient phase of NF-κB activation that is mediated by the quick degradation and subsequent recovery of IκBα, which is followed by a persistent phase of activation mediated by the slower, long-term degradation of IκBβ (211). A large body of literature has demonstrated that ROS play an essential role in the activation of NF-κB (212). Unlike the function of eNOS-derived NO, iNOS-derived NO is predominately produced by immunologically or chemically activated macrophages, as a function of an immune reaction to kill microorganisms and nitrosylates macromolecules. iNOS generates 100-1000-fold more NO than does eNOS (213). The local release of large amounts of "inflammatory NO" is often concomitantly generated with large amounts of superoxide anion from the same cell or adjacent cells, thus resulting in peroxynitrite (ONOO-) production, which mediates the cytotoxic effects in the vasculature, such as DNA damage, lipid and protein oxidation, which contribute to the pathogenesis of atherosclerosis, MI and heart failure. The presence of iNOS mRNA and protein expression has been detected in macrophages, endothelial cells and smooth muscle cells of atherosclerotic human plaques (214). iNOS expression has been found not only in advanced stages of plaques, but also in the fatty streak stage, suggesting that iNOS plays an important role in both initiating and 33  promoting atherogenesis (215; 216). iNOS expression colocalizes with oxidized lipid and nitrotyrosine (a marker for peroxynitrite production) in advanced atherosclerosis, and the colocalization suggests that a vicious pathogenic environment is promoted by the enhancing of oxidative and nitrosative stress (217). A genetic deficiency of iNOS was shown to produce decreased atherogenesis, supporting the role of iNOS in atherogenesis (218).  1.1.5.3 CD36 and Cardiovascular Dysfunction  CD36 is a member of the class B scavenger receptor family, and functions as a scavenger receptor for oxLDL (219). CD36 can be expressed in a wide variety of cells and tissues, including macrophages, adipocytes, and striated muscle cells (220). CD36 is regulated by proatherogenic cytokines and lipids, and its levels also increase after exposure of cells to LDL and oxLDL (221). The positive feedback of CD36 expression from oxLDL results in the potential for continued lipid accumulation and foam cell formation, which is a characteristic component of atherosclerotic lesions and a major pathogenic event in the development of atherosclerosis. oxLDL has high affinity for CD36 (222) and evidence shows that 65-90% of the binding and uptake of oxLDL is CD36-dependent (222; 223). A high level of CD36 expression was found in macrophages/foam cells in human atherosclerotic lesions (223; 224). Macrophages, derived from mice lacking the CD36 scavenger receptor, demonstrate reduced uptake of modified LDL (225) and reduced atherosclerosis when crossed with the ApoE mouse (222). CD36 and ApoE double knockout mice show a profound (80%) decrease in atherosclerotic lesions compared to those seen in ApoE knockout mice (222), supporting the critical role of scavenger receptors in atherogenesis.  34  1.1.5.4 Peroxynitrite and Cardiovascular Dysfunction  Peroxynitrite is produced by the reaction of superoxide anion with NO. Generally, superoxide dismutase (SOD) is the major scavenger for removing superoxide. At normal physiological conditions, concentrations of NO that cause vasorelaxation are in the order of 5-10 nM, and do not effectively compete with SOD for superoxide to make peroxynitrite. Under pathophysiological conditions; however, when iNOS is up-regulated, iNOS can produce substantially higher concentrations of NO, which rise to micromolar concentrations and can effectively compete with SOD, since the reaction between superoxide and NO is 7,000-times faster than the reaction of SOD reaction with superoxide.  L-arginine iNOS  NO  +  O2 -  citrulline ONOO-  Caspase activation  Lipid peroxidation Protein oxidation DNA oxidation  Apoptosis Necrosis  Figure 1.10 Peroxynitrite (ONOO-) induced apoptotic and necrotic cell death  35  Peroxynitrite is a well-known culprit causing endothelial injury and many other cardiovascular dysfunctions. The major mechanisms of endothelial injury associated with peroxynitrite include suppression of the production of NO and induction of endothelial cell apoptosis (186-189). Peroxynitrite and its secondary radicals can lead to various oxidative damage to proteins, lipids and DNA, resulting in inactivated metabolic enzymes, ionic pumps, and structural proteins, disrupted cell membranes, and damaged nucleic acids, ultimately leading to the dysfunction of multiple cellular processes and the induction of cell death through both apoptosis and necrosis (228) (Fig 1.10).  1.1.5.5 PM10 Exposure and Oxidative Stress Oxidative stress refers to a condition in which levels of free radicals or reactive oxygen/nitrogen species (e.g., O2–, H2O2, ONOO–) are higher than normal. ROS are capable of exerting many adverse biological effects, such as lipid/protein/DNA oxidation, and initiation of proinflammatory cascades. Oxidative stress is often induced by, and elicits inflammatory processes, and the two processes are biologically linked. iNOS, CD36, and peroxynitrite are important mediators of the ROS-regulated pathways. Leukocytes are major producers of ROS, secreted at sites of inflammation, when they phagocytose ambient particles. In addition, transition metals attached on the surface of ambient particles also contribute to ROS production (229-231). The relative importance of these two potential pathogenic factors has not been elucidated. It is believed that the induction of ROS rises first at the particle surface, and is then augmented by oxidants generated by recruited inflammatory leukocytes. The mechanisms underlying the generation of oxidative stress after exposure to PM have also not been clarified. Using an oxidant indicator dye, dichlorofluorescein, Donaldson and colleagues showed that ultrafine carbon black particles had much more surface free radical activity, compared to non-ultrafine 36  carbon black particles, suggesting a direct generation of oxidative stress at the particle surface (232). Moreover, PM10-modulated alteration of intracellular Ca2+ concentration ([Ca2+]i) has been suggested to be an additional mechanistic pathway mediating ROS generation (300; 301). PM10 and ultrafine carbon black induced rapid increases in [Ca2+]i irrespective of the presence of metal chelators. Moreover, the inhibition of Ca2+ entry was associated with reduced levels of IL-6, IL-8, and TNFα transcription (230; 303). Support for a Ca2+modulated mechanism for ROS production was further strengthened by studies in both rat alveolar macrophages and human blood monocytes showing that UFPs stimulated the opening of voltage-gated Ca2+ channels, which consequently led to modifications in various oxidants via Ca2+-mediated transcription factor activation (304; 305). Ca2+ is a ubiquitous cellular second messenger that mediates a vast array of cellular processes. Elevation of intracellular Ca2+ activates signaling cascades that are able to target the nucleus, where they modify gene transcription via the activation of transcription factors such as NF-κB and the nuclear factor of activated T cells (306). Recent studies demonstrated that noncytotoxic doses of ultrafine carbon black particles and ultrafine latex particles induce alterations in Ca2+ signaling in both human monocytic cell lines and rat bronchoalveolar lavage cells (> 85% macrophages) (232). The data suggest that, in the presence of a second stimulus: for example, a proinflammatory mediator, UFPs can have a substantial effect on intracellular Ca2+ signaling pathways and, potentially, on the expression of proinflammatory genes. Moreover, susceptible individuals, including those with pre-existing inflammation, may be more responsive to ambient particle exposure because they are already primed for Ca2+ stimulation by the cytokines in the inflammatory milieu (238). Therefore, a Ca2+modulated signaling cascade could amplify and/or propagate the deleterious effects associated with ambient particles via ROS-dependent pathways. 37  Owing to their biological activity, ROS have the propensity to exacerbate cardiovascular endpoint interactions with lipids, proteins, and DNA. For instance, ROS stimulates lipid peroxidation to produce oxLDL, which is a stimulus for monocyte migration into the subendothelial space, an early step of atherogenesis (140; 239). Human CRP is able to acquire the ability to augment platelet reactivity when treated with a transition metalascorbate system that generates reactive oxygen intermediates (240). Moreover, a link has been found between oxidative stress and the potentiation of inflammation via activation of oxidative stress-responsive transcription factors such as NF-κB and activator protein 1, which control proinflammatory genes via redox changes (241). Furthermore, the generation of oxidative stress, especially via depletion of reduced glutathione, can increase the permeability of the lung epithelium (242), allowing for the passage of particles and particleloaded macrophages into the interstitium and possibly to the circulation. Subsequently, particles could gain access to endothelial cells, the blood, and possibly be transported to other organs, though little evidence supports this hypothesis at the present.  1.1.5.6 Cyclooxygenase (COX) and Cardiovascular Dysfunction  Despite the wide use of non-steroidal anti-inflammatory drugs (NSAIDs) over the last century, the mechanism of NSAID action was not fully appreciated until 1971, when Vane published his seminal observations proposing that the ability of NSAIDs to suppress inflammation is primarily attributable to their ability to inhibit the cyclooxygenase (COX) enzyme (243). COX (PGH synthase) is the key enzyme required for the conversion of arachidonic acid to prostaglandin G2 (PHG2) and PGH2, which are subsequently converted to a variety of eicosanoids, including PGE2, PGD2, PGF2a, PGI2, and thromboxane A2 (TXA2). The array of PGs produced varies depending on the downstream enzymatic machinery present in 38  a particular cell type. For example, endothelial cells primarily produce PGI2, whereas platelets mainly produce TXA2. Eicosanoids are critical regulators of several pathophysiological responses, including inflammation, blood clotting, wound healing, blood vessel tone, and immune responses. Two COX isoforms have been identified, referred to as COX-1 and COX-2. In general, the COX-1 enzyme is produced constitutively by nearly all normal tissues (244), whereas COX-2 is inducible (e.g., at sites of inflammation), and the expression of COX-1 is low or undetectable under physiological conditions, except in the brain and kidney, where COX-2 is constitutively expressed. COX-1 is a constitutive enzyme with its expression regulated developmentally. COX1 produces prostaglandins in the endoplasmic reticulum, and these prostaglandins mediate many physiological responses via G protein-linked receptors (245). Prostaglandins, produced by COX-1, are responsible for the regulation of renal and sodium reabsorption, gastroprotection in the stomach, and vascular homeostasis. One important site of COX-1 function is at the platelet, where the enzyme is responsible for providing precursors for thromboxane synthesis, which are responsible for platelet aggregation and thrombogenesis (246). In endothelial cells, prostaglandins play a different role. The release of eicosanoids by activated platelets provides both a substrate and stimulus for the generation of prostacyclin (PGI2) by the endothelium. PGI2 stimulates vasodilatation, counteracting the vasoconstrictor, thromboxane. In contrast to COX-1, COX-2 is an inducible enzyme, similar to iNOS, which is normally absent from tissues, but is expressed in response to a wide range of extracellular and intracellular stimuli, such as growth factors, tumour promoters, or cytokines (IL-1β and TNF-α) (245). COX-2 is expressed by cells that are involved in inflammation (e.g., macrophages and monocytes); thus it has emerged as the isoform primarily responsible for 39  the synthesis of the prostanoids involved in pathological processes, such as acute and chronic inflammatory states. The promoter of the COX-2 gene contains binding sites for several transcription factors, including NF-κB, the nuclear factor for IL-6 expression (NF-IL-6), and the cyclic-AMP response element binding protein (247). Although COX-1 is mainly responsible for the biosynthesis of prostaglandins involved in homeostatic regulation, and COX-2 is primarily involved in producing prostaglandins in response to a wide spectrum of environmental insults and internal stimuli, the expression of both COX-1 and COX-2 have been detected in human atherosclerotic lesions (248). In symptomatic patients, the expression of COX-2 in atherosclerotic plaque is higher than that in asymptomatic patients (249). In addition, COX-2 knockout mice showed a 51% reduction in atherosclerotic lesions (250), and COX-2 inhibition had beneficial effects in patients with coronary artery disease by improving endothelium-dependent vasodilation and reducing lowgrade chronic inflammation and oxidative stress (251).  40  1.2 Objective, Hypothesis, and Specific Aims  1.2.1 PM10 and Cardiovascular Diseases Abundant epidemiological and experimental studies have consistently identified PM10 as a new and independent risk factor (in addition to smoking, hypertension, hyperlipidemia and diabetes) for developing and exacerbating of cardiovascular diseases. The risk of death from cardiac causes is much higher than from all other causes and significantly greater than from lung disease, which has been traditionally considered as the major adverse outcome of ambient pollutant exposure (77; 252; 253). Reductions in PM10 levels are linked with improvements in health outcomes (6; 27; 38; 254-255), and these findings not only indicate a causative effect of exposure to PM10 and health consequences, but also imply that PM10 effects are partly reversible. Increased levels of ambient PM, as a consequence of rapid industrialization and urbanization, have become a major concern of public health professionals and the governments around the world. Positive steps have been taken by the governments to prevent and reduce ambient pollution since the London fog disaster in 1952. Nevertheless, a permanent reduction in urban ambient particles needs time. Because of its ubiquitous and involuntary nature, exposure to PM10 may continuously enhance the cardiovascular risk among millions of susceptible people worldwide in an often inconspicuous manner. Beyond serving as a simple trigger, exposure to PM10 can also elicit numerous adverse biological responses (such as systemic inflammation) that may further augment cardiovascular risk over the long term after months to years of exposure. Therefore, understanding the biological signaling pathways and then initiating medical prevention could be an immediate and effective strategy for alleviating PM10-induced cardiovascular mortality and morbidity. Since the early 1990’s, numerous researchers and clinicians have vigorously worked on delineating 41  the signaling pathways mediating exposure to PM10-augmented cardiovascular morbidity and mortality; yet, the way in which PM10 negatively influences cardiovascular function has not been well understood. In any case, two hypotheses have been proposed. The majority of studies suggest that inhaled ambient PM provokes an inflammatory response in the lung, which causes lung inflammation with consequent release of prothrombotic and inflammatory cytokines into the circulation (81; 103; 104). Meanwhile lung inflammation stimulates the release of bone-marrow-derived neutrophils and monocytes that contribute to the secondary cardiovascular effects.  1.2.2 Shortcomings of Published Studies Population and experimental-based studies have provided some insights into the mechanisms whereby exposure to PM10 promotes cardiovascular abnormalities, including endothelial dysfunction and increased blood pressure (22). Nevertheless, epidemiological studies, by their very nature, provide only a partial view of causative mechanisms. In addition, these epidemiological data are limited by imprecise measurements of the ambient particle levels. The evidence from mechanismic investigations is mainly from in vitro models or from a simplified approach (instillation model) injecting PM10 collected from filters or surrogate particles into the trachea. Despite several advantages of the instillation model, questions arise about how well instillation exposure can represent the inhalation in the real world. In addition, the recovery of particles on filters is variable, and a question may be asked whether or not all components can be retrieved in quantities that reflect the original particles. In this dissertation, we conducted a study in a diesel exhaust (DE) exposure facility at the University of Washington, and overcame many shortcomings of the in vitro and instillation experiments. The facility consists of a DE exposure system, which incorporates a 42  diesel-electric generator for producing diesel exhaust. DE was sampled from the generator exhaust pipe, and then delivered to a dilution/ventilation system, where the concentrations were controlled and monitored. In addition, the temperature and humidity of the DE were conditioned and maintained before being conveyed to the animal exposure chambers.  1.2.3 Diesel Exhaust (DE) Mounting evidence has consistently shown that traffic-derived pollution is strongly associated with increased cardiovascular diseases (256-258). The analyses of many cohort studies from different countries, including the United States (259), Germany (145), the United Kingdom (15), Canada (260), and the Netherlands (261), indicate that high traffic density and living near major roads are associated with increased cardiovascular mortality. Chronic exposure to traffic-generated PM has been linked with an increased risk of fatal MI in Sweden (262). Recently, an analysis from a cohort in the Netherlands demonstrated that traffic intensity was associated with cardiovascular mortality with the highest relative risk for ischemic heart disease after adjusting for higher levels of traffic noise (263). Another population-based study, with 4,494 subjects from Germany found that residential proximity to major roadways was associated with increased coronary atherosclerosis (145). Of the traffic-related air pollution, DE is the single biggest contributor, accounting for up to 90% of the total particles in some cities.  1.2.4 Objective, Hpothesis, Rationales, and Specific Aims Recent analysis of the American Cancer Society data from 1.2 million adults residing in 50 states indicates that the largest specific cause of death associated with exposure to ambient PM is ischaemic heart disease, which accounts for 25% of all causes (40). Atherosclerosis plays an important role in the pathogenesis of ischemic heart diseases. The development of atherosclerosis is well known to result from the chronic inflammatory 43  response (140). Studies from our laboratory have reported that intratracheal instillation of PM10 promoted the development of atherosclerosis in Watanabe heritable hyperlipidemic rabbits (141). Nevertheless, no study using an inhalation model has demonstrated whether or not exposure to DE causes atherogenesis. Thus, the overall objective of this work is to examine the impact of DE inhalation on the progression of atherosclerosis, and to investigate mechanismtic links between DE exposure and atherogenesis.  1.2.4.1 Rationale for Specific Aim 1  ROS is known to be implicated in the development of atherosclerosis; and some human studies have directly investigated the occurrence of systemic oxidative stress in relation to DE, or traffic-related exposure (264). Bräuner and colleagues recently reported that the exposure to ambient urban particles derived from traffic led to DNA oxidation in healthy young adults (264). Studies of young adults, conducted in Denmark, also showed elevations in biomarkers of protein, lipid, and DNA oxidation induced by traffic-derived ambient particle exposure (265-267). Nevertheless, the morphological modifications of atherosclerostic plaques induced by DE inhalation have not been examined. Specific Aim 1: Assess the impact of DE inhalation on atherogenesis by quantifying the compositional changes of atherosclerotic plaques, and relate these changes to the level of systemic oxidative stress.  1.2.4.2 Rationale for Specific Aim 2  Inducible nitric oxide synthase (iNOS) is up-regulated in response to inflammatory cytokines as part of the host defense responses (268), and can generate 100–1000-fold more nitric oxide (NO) than can eNOS. The large amount of locally released NO has been linked to the generation of harmful oxidative products, such as peroxynitrite, which is implicated in 44  the iNOS-mediated development of atherosclerosis (143; 186; 189). Atherosclerotic lesions were diminished in iNOS/ApoE double knockout mice (218; 269), suggesting that iNOS plays an important role in atherogenesis. iNOS over-expression was shown to be responsible for DEP-induced lung inflammation (186; 270), while iNOS knockout mice had a significant reduction of cytokines in the lung after exposure to ambient particles (271). Sun and colleagues recently reported that exposure ApoE knockout mice to concentrated ambient particles for six months increased the iNOS expression in the aortic root (143). The potential role of iNOS in the progression of atherosclerosis induced by exposure to DE; however, is still unknown. Specific Aim 2: Examine the potential role of iNOS in atherogenesis induced by DE exposure.  1.2.4.3 Rationale for Specific Aim 3  Prostanoids play an imperative role in cardiovascular function, such as in regulating vascular tone, modulating the inflammatory response, control leukocyte-endothelial cell adhesion and in platelet aggregation (272). Cyclooxygenase (COX) is the key regulatory enzyme responsible for the formation of prostanoids. Two COX isozymes, COX-1 and COX-2, have been characterized, and implicated in cardiovascular diseases, such as atherosclerosis (250; 273). In vitro studies showed that exposure of PM10 to human airway epithelial cells induced overexpression of COX-2, which led to lung inflammation and injury (274). It was also reported that incubation of DE with human macrophage cells increased COX-2 protein and mRNA expression, which were associated with a subsequent increase in cholesterol accumulation and foam cell formation (275). Nevertheless, whether or not DE inhalation modifies COX-1 and/or COX-2 activity and expression in blood vessels has not been investigated. 45  Specific Aim 3: Determine the impact of DE inhalation on COX function and expression in blood vessels.  1.2.4.4 Rationales for Specific Aim 4  The endothelium, a monolayer of cells separating blood from the vascular wall, serves as an important regulator to maintain vascular tone and vasculature integrity (276-278). It does so by producing a number of mediators, in particular, nitric oxide (NO) and endothelin (ET). Endothelium-derived NO prevents blood vessels from developing atherosclerosis (279). Many in vitro and ex vivo experiments have demonstrated the capacity of DE attenuated NO-dependent dilation, which was mediated via proinflammatory and ROS-dependent mechanisms (183; 280-282). In contrast to NO, ET is a vasoconstrictor and implicated in the pathogenesis of atherosclerosis (283). Exposure to PM10 causes excessive ET-1 production (196), increased vascular sensitivity to ET-1 (284), and up-regulation of ET-1 mRNA expression (200), which results in exaggerated vasoconstriction and could contribute to vascular events such as MI and stroke. In any case, whether or not exposure to DE causes endothelial dysfunction, which could contribute to atherogenesis, is unknown. Specific Aim 4: Assess the effects of exposure to DE on NO-mediated vascular endothelial function.  46  Chapter 2 Methodology  2.1 Exposure Protocol and Experimental Animals  2.1.1 DE Exposure System DE was derived from a 2002 model turbocharged direct-injection 5.9 liter Cummins B-series engine (6BT5.9G6, Cummins, Inc., Columbus, IN) in a 100 kW generator set located just outside the laboratory. This engine is comparable to that used in heavy-duty road applications such as delivery trucks and school buses. Fuel is #2 un-dyed on-highway fuel from a commercial source that was remain stable throughout the experiments. Load is maintained at 75% of rated capacity, using a load-adjusting load bank (Simplex, Springfield, IL), throughout the experiments. All dilution air for the system was passed through HEPA, and carbon filters, permitting a filtered air control exposure option with very low particulate and gaseous organic pollutant levels (Table 2.1). The air entering the exposure room was conditioned to 18°C and 60% relative humidity. During the exposures, DE concentrations were continuously measured. Multistage samples were collected on a micro-orifice uniform deposition impactor (MOUDI; MSP, Shoreview, MN) which indicated a mass median diameter of 0.104 µm. The aerosol number-size distribution between 40nm and 2.5µm aerodynamic diameter were measured with a differential mobility analyzer (DMA), condensation particle counter sensor (CPC, 40 to 600nm) and an aerodynamic particle counter (APS, 500nm to 10µm). The two CPCs (one with 100 nm diffusion screen) provide assurance that the particle size distribution did not change between the intensive 47  characterization procedures. This measurement of the absolute number concentration as a function of particle size was made with 15-minute time resolution. Several gas phase chemicals (CO, CO2, NO, NO2, water vapor and hydrocarbon species) were monitored continuously using Fourier-Transformed Infrared Technology (FTIR). The maximum 24-hour PM10 concentrations reported by US Environmental Protection Agency (EPA) range from 26 to 534 µg/m3 (US Environmental Protection Agency: http://www.epa.gov/airtrends/msafactbook-db.pdf.). The exposure level (200µg/m3) in this study was determined to reflect the ambient PM level of a heavy traffic condition. The average calculated exposure throughout a 24-hour period in our study was less than 35 µg/m3, which is within the range of the National Ambient Air Quality Standard (285) and is environmentally relevant (15). 2.1.2. Animals and Exposure Protocol Male ApoE knockout mice were housed in a temperature- and humidity-controlled environment with a 12-h light/dark cycle with free access to water, and standard rodent chow diet (Harlan Teklad). At the age of 30 weeks, these mice were moved to a “Biozone” facility adjacent to the exposure chamber where exposure was controlled by opening or closing a valve to animal cages resulting in minimal stress for animals during the exposure period. ApoE knockout mice (24/group) were randomly chosen and exposed for 7 weeks (5days/week, 6hrs/day) to DE at the concentration of 200 µg/m3 particulate matter. Mice exposed to filtered air were the control. Animal procedures were approved by the Animal Care and Use Committee of the University of Washington.  2.2 Sample Collection  After exposure, sodium pentobarbital (100mg/kg, Abbott Laboratories, IL) and heparin sulfate (500U/kg) were administered intraperitoneally. Upon the loss of all reflexes, 48  blood was collected from inferior vena cava and put into EDTA tubes. Plasma was obtained after centrifugation of blood and stored at –80°C until assay. Urine was collected and kept at –80°C until assay. The thoracic aorta, aortic root, and the lung were carefully dissected from their connective tissues and kept in appropriate conditions until assay. The thoracic aorta was harvested and preserved in three ways for different analysis: 1) fresh tissue (~4mm) for vascular function study using wire myograph; 2) part of the thoracic aorta (~5mm) was embedded in OCT (Sakura Finetek), rapidly frozen on dry ice, and kept at –80°C; 3) the rest of the thoracic aorta (~5mm) was fixed with 10% neutral formalin for 24 hours, then embedded in paraffin. Some of the aortic roots were fixed with 10% neutral formalin for 24 hours, then embedded in paraffin, and some of the roots were in OCT and rapidly frozen on dry ice, then kept at –80°C. Table 2.1 Composition of Diesel Exhaust Particles (DEP) generated at the Northlake exposure facility.  Filtered Air 12.99 PM2.5(µg/m3)* CO(ppm) 0.28 NO(ppb) 38.63 NO2(ppb) 15.50 3 Particle #/cm 600 * Measured in the mouse cages.  DEP 187.71 0.76 1515.80 25.56 60,000  2.3 Peripheral Band Cell Count  To examine the systemic inflammatory response, the peripheral band cell count was performed. Blood smears were fixed with methanol and stained with Wright staining (Sigma Chemicals). A hundred PMNs from randomly selected fields were manually counted and the percentage of band cells was determined.  49  2.4 Tissue Processing and Image Acquisition  Before staining, paraffin-embedded blood vessel sections were cut into 5µm thick section, deparaffinized in xylene and hydrated by passing through a series of graded alcohol, and cryosections (5µm) were air-dry for 1h, fixed with cold (4°C) acetone for 15min. After the sections were stained for specific targets as explained in details in the following sections, images were captured by a spot digital camera (Microspot, Nikon, Tokyo, Japan), coded and examined without knowledge of the experimental groups. The size of an atherosclerotic plaque, and its compositions were determined by quantitative a morphologic analysis explained in detailed in the following sections.  2.5 Morphometric Analysis of the Lung Tissue and the Composition of Atherosclerotic Plaque  2.5.1 Lung Tissue Lungs were inflated via the trachea with 10% neutral formalin for 24 hours, cut into 4 longitudinal slices, embedded in paraffin blocks, sectioned at 5µm and stained with hematoxylin and eosin (H&E). The images were captured, coded, and analyzed using point counting method. Briefly, a grid of points was superimposed onto the captured images by Image Pro Plus software. The density of the grid, and the number of fields were selected to maintain the coefficient of error of the estimate of the volume below 0.1. The points that had contact with alveolar macrophages were counted. The volume fraction (V/v %) of alveolar macrophages was obtained by normalizing the total points on alveolar macrophages to the total points on the lung alveoli. In addition, 100 alveolar macrophages were randomly selected in these lung sections (4 views/sections total 16 views/mouse), and the number of alveolar macrophages positive for particles was enumerated. 50  2.5.2 Atheroslcerotic Lesions in Aortic Root 2.5.2.1 Lesion Size, Foam Cells, and Cellularity  Sections that contained three complete valve leaflets were stained with Movat pentachrome to quantify atherosclerotic lesion area, total cell counts, and foam cells. To identify foam cells, we stained the aortic root sections with rat monoclonal anti F4/80 antibody (1:50, AbD Serotec), a specific marker for mature mouse macrophages. Some of the foam cells were not stained with the F4/80 antibody, whereas some of these stained cells did not contain any lipid in their cytoplasm. Therefore, we used a combination of positive staining for F4/80 antibody, and Movat pentachrome staining, where foam cells or macrophages contain lipid in their cytoplasm to identify foam cells. The images were captured, coded, and analyzed using a grid of points superimposed onto the captured images by Image Pro Plus software. The volume fraction (V/v %) of atherosclerotic lesions was obtained by counting the total points falling in the atherosclerotic lesions divided by the total points on the valve leaflets area. Using the same method, total points for foam cells in atherosclerotic lesions were counted and normalized to the total points in the lesion area to obtain the volume fraction of foam cells in plaques. The nuclei of all cells in atherosclerotic lesions were also counted and normalized to the size of atherosclerotic lesion to obtain the volume fraction of cellularity.  2.5.2.2 Lipid Content of Plaque  Frozen aortic roots embedded in OCT (Sakura Finetek) were cut 5µm thick by a microtome. Sections that contained three complete valve leaflets were fixed in 4% paraformaldehyde at room temperature for 30 min, and stained with Oil-Red-O (Sigma Chemicals) to identify lipid in the plaques. The area of positive staining was quantified by 51  colour segmentation using Image Pro Plus software, and normalized to the area of atherosclerotic lesion to obtain the volume fraction (V/v %) of positive staining which represents the lipid.  2.5.2.2 Collagen Content of Plaque  Aortic root sections that contained three complete valve leaflets were stained in picrosirius red (Sigma Chemicals) for one hour, washed in two changes of acidified water, dehydrated in three changes of 100% ethanol. Using Image Pro Plus software, the area of red staining was recognized, quantified by colour segmentation, and normalized to the area of the atherosclerotic lesion to obtain the volume fraction (V/v %) of positive staining for picrosirius red, representing collagen content.  2.6 Vascular Function Study  Thoracic aortae were carefully cleaned off connective tissue without damaging the endothelium, and placed in ice-cold physiological salt solution (PSS). The vessels were cut to 2mm rings and mounted on a wire myograph (Model 610M; Danish Myo Technology, Aarhus, Denmark). Each vessel was bathed in oxygenated PSS at 37°C for an hour during which the resting tension was gradually increased to 6mN with three changes of PSS at 10 minute intervals followed by stabilizing the vessels at resting tension (6mN) for 30min. Thereafter, the vessels were stimulated with 80 mM KCl twice. Smooth muscle contractility was studied by the addition of cumulative concentrations of phenylephrine (PE, 1nM-10µM). To examine the impact of DE on iNOS activity, 1400W, a specific inhibitor for inducible NO synthase (iNOS), was used. To access the effect of DE exposure on cyclooxygenase (COX), indomechacin (1µM) was applied. To examine the specific roles of COX1 and COX2, SC560 (10µM) and NS398 (10µM) were used to inhibit COX1 and COX2, 52  respectively. To examine the impact of DE exposure on the ET-1 production, the ET receptor antagonist (bosentan, 10µM), the selective inhibitors for ETA (BQ123, 10µM), and ETB (BQ788, 10µM) were administered, respectively. PE (1nM-10µM) dose-response curves were conducted before and after incubating with these antagonists, respectively. Once a sustained constriction was evoked, cumulative concentrations of ACh (1nM10µM) or SNP (1nM-10µM) were applied to evaluate endothelium-dependent or independent NO-mediated vasodilation, respectively. To examine the effect of DE exposure on the endogenous NO production, the vessels were incubated with NG-nitro-L-argininemethyl ester (L-NAME; 200µM), a NO synthase (NOS) inhibitor.  2.7 Immunohistochemical Analysis of Macrophages (F4/80), iNOS, CD36, Nitrotyrosine, α-actin, COX-1, COX-2, eNOS, and sGC Expression  Paraffin-embedded thoracic aorta and aortic root sections that contained three complete valve leaflets were deparaffinized in xylene and hydrated by passing through a series of graded alcohol. Citrate buffer (Invitrogen) was used for antigen retrieval. Sections were then incubated with 10% goat serum at room temperature for 30min to block nonspecific binding proteins. Aortic root sections were respectively incubated with these antibodies: rabbit polyclonal biotinylated antibodies to inducible nitric oxide synthase (iNOS) (1:100, Santa Cruz); CD36 (1:50, Santa Cruz); nitrotyrosine (NT) (1:400, Upstate Biotechnology), α-actin (1:600, Abcam), COX-1 (1:150, Cayman Chemical), COX-2 (1:100, Santa Cruz), and rat monoclonal biotinylated anti F4/80 antibody (1:50, AbD Serotec) at 4°C overnight. Thoracic aorta sections were incubated with rabbit polyclonal antibodies to COX1 (1:150, Cayman Chemical), COX-2 (1:100, Santa Cruz), sGCα1 (1:200, Abcam) and sGCβ1 (1:200, Abcam) at 4°C overnight. Negative controls were included with non-immune isotype antibody, or omission of the primary antibody. Subsequently, sections were 53  incubated with biotinylated goat anti rabbit IgG (1:800, Vector Laboratories) at room temperature for 30min, followed by avidin-biotin conjugated alkaline phosphatase, and Vector red (Vector Laboratories) to detect the antigen-antibody complexes. To detect the eNOS expression in the thoracic aorta, cryosections (5µm) were air-dry for 1h, fixed with cold (4°C) acetone for 15min. M.O.M. kit (Vector Laboratories) was used to block non-specific binding proteins following the manufacture’s instruction. Thereafter, sections were incubated with monoclonal anti-eNOS antibody (1:50, BD Transduction Laboratories) at room temperature for 1h, followed by M.O.M biotinylated anti-mouse IgG (Vector Laboratories) at room temperature for 10min, then incubated by avidin-biotin conjugated alkaline phosphatase and Vector red (Vector Laboratories) to detect the antigenantibody complexes. The positive red staining was recognized, quantified by colour segmentation analysis using Image Pro Plus software, and normalized to the area of the atherosclerotic lesion in aortic roots, or normalized to the thickness of the vascular wall for the sGC expression, or normalized to the length of the intima for the eNOS expression, to obtain the volume fraction of each specific protein expression.  2.8 Western Immunoblotting Analysis of iNOS Expression  Due to limited quantity of vessel samples, the heart tissues were used to evaluate the iNOS expression by western immunoblotting. The frozen heart tissues were homogenized in 10 volumes of ice-cold RIPA buffer (Sigma Chemicals) supplemented with protease inhibitor cocktail (Roche Diagnostics). Supernatant was obtained following centrifugation of homogenates at 12,000 rpm for 15 min, and the protein concentration was quantified by Bradford method using Bio-Rad DC Protein Assay kit according to the manufacturer’s instructions (Biorad). Protein samples (40µg/lane) were separated on 7.5% SDS-PAGE gel, 54  subsequently electrotransferred to polyvinyldifluoride membranes (Biorad) and blocked with 5% bovine serum albumin (BSA) dissolved in 0.1% TBS-Tween 20 for 60min at room temperature. The membrane was incubated with specific primary antibody for iNOS (1:500, Santa Cruz) at 4°C overnight, followed by 1-hour incubation with horseradish peroxidase– conjugated goat anti-rabbit IgG (Santa Cruz). Immunoreactive bands were visualized by enhanced chemiluminescence kit (Amersham Life Sciences). Densitometry was carried out using Image J (NIH), and the results were expressed in optical intensity per mg protein.  2.9 ELISA of NF-κ κB (p65) Activity  Nuclear extracts from the frozen heart tissues were obtained using nuclear extract kit (Cayman Chemical). Protein concentrations of the nuclear extract were quantified by Bradford method using Bio-Rad DC Protein Assay kit according to the manufacturer’s instructions (Biorad). NF-κB (p65) activity in the nuclear extracts from heart tissues was measured by enzyme-linked immunosorbent assay (ELISA) using the NF-κB (p65) transcription factor kit (Cayman Chemical) according to the manufacturer’s instructions. This kit is a non-radioactive, sensitive method for detecting NF-κB (p65) DNA binding activity in nuclear extracts as a replacement for the radioactive electrophoretic mobility shift assay. Results were expressed as absorbance (450nm) per mg protein.  2.10 Assessment of Systemic Oxidative Stress  It has been reported that isoprostanes are stable end products of arachidonic acid peroxidation (391). Of the variety of isoprostanes detected, 15-F2t-isoprostane (15-F2t-IsoP) has been found to be a specific, reliable marker of lipid oxidation. Urine samples were purified using 8-isoprostane affinity sorbent, and the levels of 15-F2t-IsoP in urine were 55  measured using 8-Isoprostane EIA kit according to the manufacturer’s instruction (Cayman Chemical). Urine creatinine levels were measured by creatinine assay kit (Cayman Chemical). The results were expressed in pg of 15-F2t-Iso per mmol creatinine. 8-hydroxy-2-deoxy guanosine (8-OH-dG) is produced by oxidative damage of DNA due to the production of reactive oxygen and nitrogen species, and has been served as an established marker for DNA oxidation. Because of the complexity of plasma samples, urine is more suitable for measuring free 8-OH-dG. 8-OH-dG concentration was measured using 8-OH-dG kit (Cayman Chemical). The urine creatinine levels were measured by creatinine asssy kit (Cayman Chemical). The results were expressed in pg of 8-OH-dG per mmol creatinine.  2.11 Measurement of Urine 2,3-dinor-6-keto prostaglandin F1α α  2,3-dinor-6-keto prostaglandin F1α is a major urinary metabolite converted from PGI2. PGI2 level was assessed by measuring urine 2,3-dinor-6-keto prostaglandin F1α using an EIA kit according to the manufacturer’s instruction (Cayman Chemical). The urine creatinine levels were measured by creatinine asssy kit (Cayman Chemical). The results were normalized to urine creatinine levels and expressed in pg/ml PGI2 per mg/dl creatinine.  2.12 Measurement of Plasma Nitrite and Urine cGMP  Under physiological conditions, within a few seconds, NO is oxidized to nitrites or nitrates, which are stable metabolites of NO. Plasma NO levels were assessed by measuring nitrite levels using a NO-specific chemiluminescence analyzer (Sievers, Boulder). cGMP was measured in urine using cGMP EIA kit according to the manufacturer’s instruction (Cayman Chemical). Creatinine levels were measured by creatinine assay kit (Cayman Chemical). 56  2.13 Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) of mRNA Expression of iNOS, NT, NFκ κB, COX1, COX2 and PGI2 in the Heart, and mRNA Expression of eNOS and sGCα α in the Abodominal Aorta  Total RNA was extracted from the heart tissues or the abdominal aortae (for eNOS, and sGC) using RNeasy Fibrous Tissue Mini Kit (Qiagen). RNA concentration was measured using Nanodrop (Thermo Scientific). Reverse transcription was performed using a RT kit (Invitrogen). The same amount of RNA was loaded in triplicates for each assay, and RT-PCR was performed using TaqMan Universal PCR master mix (Applied Biosystems) according to the manufacturer’s instructions. The mRNA expression of iNOS, NT, NFκB, COX1, COX2, PGI2, eNOS, sGC, β-actin, and hypoxanthine phosphoribosyltransferase-1 (HPRT1) was measured by qRT-PCR (TaqMan) using the ABI Prism 7900HT sequence detection system (Applied Biosystems, 40 cycles consisting of 15s at 95°C and 1min. at 60°C). The values of gene expression were calculated based on the comparative threshold cycle (Ct) method, normalized to the expression values of β-actin and HPRT1, and displayed as ratio relative to β-actin.  2.14 Solutions and Chemicals  The PSS consisted of the following (in mM): NaCl 119, KCl 4.7, KH2PO4 1.18, NaHCO3 24, MgSO47H2O 1.17, CaCl2 1.6, glucose 5.5 and EDTA 0.026. All reagents were purchased from Sigma Chemicals unless specifically mentioned.  2.15 Statistical Analysis  Results are reported as mean±SEM. The statistical significance was evaluated using the Student’s t test for unpaired values of two groups. The concentration-response curves of 57  the different groups were compared by ANOVA for repeated measurements followed by Bonferroni’s correction. P<0.05 was considered to be significant. In all experiments, n equals the number of mice from which samples were obtained.  58  Chapter 3 Changes in Atherosclerotic Plaques Induced by Inhalation of Diesel Exhaust1  3.1 Introduction  Epidemiological studies conducted over the past 20 years showed that exposure to ambient particulate matter air pollution with aerodynamic diameter less than 2.5µm (PM2.5) maybe an independent risk factor for increased cardiovascular morbidity and mortality (22; 257), and recent studies suggest that reducing ambient particles resulted in declined cardiovascular morbidity and deaths (37; 38). Evidence from other and our own laboratories suggested that the deposition of particles in the lung causes alveolar inflammation resulting in a systemic inflammatory response that impacts blood vessels (5), which could be responsible for the downstream adverse cardiovascular effects (22). This hypothesis is supported by several animal studies (6-8), and human studies showing that an elevation of 10 3  3  µg/m or 20 µg/m in PM2.5 was associated with 5.9% or 12.1% increases in the development of atherosclerosis, respectively (144). Inflammation plays a central role in all stages of atherosclerosis development (398). Exposure to ambient PM results in excessive production of reactive oxidative species (ROS) (287). ROS contribute to many cardiovascular complications via exacerbating interactions with lipids, proteins, and DNA. For instance, in vitro study showed that diesel exhaust --------------------------------------------------------------------------------------------------------------1  A modified version (with more detailed Methods) of this chapter has been published. Ni Bai, Takashi Kido, Hisashi Suzuki, Grace Yang, Terrance J. Kavanagh, Joel D. Kaufman, Michael E. Rosenfeld, Cornelis van Breemen, Stephan F. van Eeden (2011). Atherosclerosis.  59  particles (DEP)-induced ROS exerted lipid peroxidation to produce oxidized low-densitylipoprotein (oxLDL), which can serve as a stimulus for monocyte migration into the subendothelial space representing an key step in atherosclerosis (140; 181). These monocytederived macrophages phagocytose oxLDL, contributing to foam cell formation, and the development of fatty streaks, a hallmark of early atherosclerotic lesion (141). Macrophage CD36 is the major scavenger receptor for oxLDL, hence promoting the progression of atherosclerosis (403). Active unstable atherosclerotic plaques are characterized by increased lipid accumulation, foam cell formation, smooth muscle migration from the sub-endothelial layer, and a thin fibrous cap (289). Plaque disruption and atherothrombosis are the underlying causes of approximately 60-80% of all sudden cardiac deaths (290). Diesel exhaust (DE) is a major component of urban PM2.5, accounting for up to 90% of the fine particulate mass in ambient air of many major cities, such as London (291). DE is a mixture of fine particles and gases, and represents a useful model of traffic-related air pollutants. The particulate from DE consists of a central carbon core nucleus onto which an estimated 18,000 combustion products are adsorbed, including organic chemicals, such as polycyclic aromatic hydrocarbons, and transition metals (19; 292). Recent studies have shown an association between progression of atherosclerosis and people living near major roads, and traffic-related air pollution exposure and acute coronary events (293). The association between DE exposure and cardiovascular diseases is compelling; however, the impact of DE exposure on atherogenesis is poorly understood. In this study, we exposed ApoE knockout mice to DE via whole-body inhalation for 7 weeks, and measured morphological alterations of atherosclerotic lesions to test the hypothesis that DE inhalation causes changes in atherosclerotic plaque characteristic of unstable plaque or plaque vulnerable to cause acute vascular events. We also explored the impact of the  60  magnitude of DE exposure, and downstream oxidative stress on changes in plaque morphology.  3.2 Methods and Materials (Please refer to Chapter 2 for details) 3.2.1 Methods Male ApoE knockout mice fed with regular chow, at the age of 30-week old, were exposed for 7 weeks (5days/week, 6hrs/day) to DE (200 µg/m3 of particulate matter). Mice exposed to filtered air were the control. After exposure, mice were euthanized, and the plasma, thoracic aorta, aortic root and lung were collected and kept at appropriate conditions until assay. Blood smears were fixed with methanol and stained with Wright staining. A hundred PMNs from randomly selected fields were manually counted and the percentage of band cells was determined. Plasma cholesterol and triglyceride levels were measured using commercially available kits. For morphometric analysis, images were captured by a spot digital camera (Microspot, Nikon, Japan), coded and analyzed using Image Pro Plus software. 10-12 fields per view for each animal were randomly chosen. Lung were inflated and fixed with 10% neutral formalin for 24 hours, sectioned at 5µm and stained with hematoxylin and eosin (H&E). Random images (16 views/mouse) were captured to represent the whole lung and analyzed using point counting to determine the volume fraction (V/v%) of alveolar macrophages. The V/v% of alveolar macrophages positive for particulate matter was measure of the magnitude of lung exposure. Aortic root was used to quantify compositional changes in plaque. Aortic root tissues were fixed with 10% neutral formalin for 24 hours, embedded in paraffin, sectioned at 5µm 61  and stained with Movat pentachrome to determine the V/v% of plaques size, total cell counts (plaque cellularity), and foam cells. The nuclei of cells in each lesion were enumerated and normalized to the plaque size. Picro-sirius red staining was used to identify collagen content in aortic root lesions. Frozen aortic root tissues were embedded in OCT, sectioned at 5µm, stained with Oil-Red-O and the V/v of positive staining was determined. The expression of iNOS, CD36, nitrotyrosine, and α-actin was quantified after immunohistochemical staining for aortic root tissues.  Sections were incubated with  antibodies F4/80, a marker for mature macrophages (1:50); inducible nitric oxide synthase (iNOS) (1:100); CD36 (1:50); nitrotyrosine (NT) (1:400), and α-actin (1:600) at 4°C overnight. Subsequently, sections were incubated with biotinylated goat anti rabbit IgG (1:800), followed by avidin- biotin conjugated alkaline phosphatase and Vector red (Vector Laboratories) to detect the antigen-antibody complexes. The area of positive staining was recognized, and the volume fraction (V/v %) of iNOS, CD36, and NT was determined. Urine samples were purified using 8-isoprostane affinity sorbent, and the concentration of 15-F2t-isoprostane (15-F2t-IsoP), a marker of lipid oxidation, was measured using a 8-Isoprostane EIA kit. As a marker of DNA oxidation, 8-hydroxy-2-deoxy guanosine (8-OH-dG) concentration was measured using a 8-OH-dG kit. Creatinine levels were measured by a creatinine assay kit. Plasma MPO levels were also measured using a myeloperoxidase (MPO) ELISA kit.  3.2.2 Statistical Analysis  Data are shown as mean±SEM. The statistical significance was evaluated using the unpaired Student’s t test for simple comparison between two values. Linear regression modeling was used to assess the relationship between exposure markers (alveolar macrophages with particles) and compositional changes in plaques and oxidative stress 62  markers. In all experiments, n equals the number of mice from which samples were obtained. P<0.05 was considered to be significant. 3.3 Results  3.3.1 Plasma Lipid To examine whether DE exposure had any effect on the lipid profile, we measured plasma cholesterol and triglyceride. The plasma cholesterol and triglyceride level were similar after exposure to DE, compared with the filtered air control (Table 3.1). Table 3.1 Plasma cholesterol and triglyceride level (Values are mean±SEM).  Cholesterol (M) Triglyceride (mg/dl)  Filtered air 26.2 ± 1.0 135.8 ± 11.8  DE 28.0 ± 1.8 140.6 ± 12.9  n=12, P=0.4 n=12, P=0.8  3.3.2 Alveolar Macrophages in the Lung Tissue DE exposure significantly increased the number of alveolar macrophages positive for particles (3.5±1.2% in Filtered air vs.85.7±1.7% in DE; p<0.0001) (Fig.3.1A). The total number of alveolar macrophages was also increased in DE exposure group, compared with filtered air exposure (1.3±0.2% vs.10.2±1.1%; Filtered air vs. DE; p<0.01) (Fig.3.1B).  63  Alveolar Macrophages Positive for Particles(%)  B  Filtered Air DE  80  *  60 40 20 0 Filtered Air  DE  Alveolar Macrophage (V/v%)  A 100  15  Filtered Air DE  *  10  5  0 Filtered Air  DE  Figure 3.1 Exposure effects. A) Exposure to DE increased the number of alveolar macrophages positive for particles, n=10, *P<0.0001; B) Total alveolar macrophages were also increased after DE exposure, n=9, *P<0.01. Values are mean±SEM.  3.3.3 Increased Circulating Band Cells and Positive Correlation with Lung Inflammation The blood smear band cell counts were significantly higher after DE exposure than the control (filtered air) (Fig.3.2A). In adition, the elevation of band cell counts was positively correlated with increased alveolar macrophages (Fig.3.2B), suggesting a strong link between lung inflammation, and the systemic response.  20 15  Filtered Air DE  B *  10 5 0  Filtered Air  DE  Alveolar Macrophage (V/v,%)  Blood Smear Band Cell Count (%)  A 20  Y=1.1927x-7.6649 R2=0.61  10  0 5 10 15 Blood Smear Band Cell Counts (%)  20  -10  Figure 3.2 Circulating band cell counts and their relationship with alveolar macrophages. A) Increased circulating band cells after DE exposure, compared with the filtered air exposure. n=12, *P<0.01;. B) Positive correlation between circulating band cells and alveolar macrophages, P=0.0045. Values are mean±SEM.  64  3.3.4 Compositional Changes in Atherosclerotic Plaque Figure 3.3A, C, and E show different morphological features of plaques on Movat stains. There was no difference in the volume fraction of the size of atherosclerotic lesions between DE and filtered air exposed mice (Fig.3.3B). Histological characterization of plaque morphology reveals that DE exposure increased plaque cellularity (70.0±4.0 vs. 100.0±9.8cells/100µm of lesion area; Filtered air vs. DE; p<0.02) (Fig.3.3D), foam cell formation (12.2±1.7% vs. 28.1±7.8%; Filtered air vs DE; p<0.04) (Fig.3.3G), increased lipid accumulation (16.1±2.1% vs. 31.9±4.7%; Filtered air vs. DE; p<0.02) (Fig.3.4A,B,C), and smooth muscle cell content (7.5±2.0% vs. 25.5±9.1%; Filtered air vs DE; p<0.05) (Fig.3.4D,E,F). We observed a trend for decreased collagen content of plaques (90.2±9.6% vs. 69.4±9.1%; Filtered air vs DE; p>0.05) (Fig.3.4G,H,I). The proteoglycan content of plaques was similar between DE and filtered air exposure groups (Appendix A).  65  Nu mber of Ce lls µ m) / Plaq ue Are a (100µ  Volume Fraction of Plaque Size (V/v %) (Nor malized to Aor tic Root Are a)  A B  60  150  Filtered Air DE  40  20  0 Filtered Air  C  Filtered Air  DE  50µm  D  20µm Filtered Air DE  100  *  50  0 DE  66  E  F  50µm  20µm  Volume F raction of Foam Cells (V/v%) (Nor malized to Plaqu e Ar ea)  G  40  Filtered Air DE  *  30 20 10 0 Filtered Air  DE  Figure 3.3 Movat staining analysis of atherosclerotic lesion size, cellularity and the number of foam cells in aortic root. A) Representative photomicrographs of Movat staining for plaque size analysis (100X); B) No changes in volume fraction of the size of aortic root lesions were observed, n=10, P>0.05; C) Movat staining for total cell count (400X); D) The cellularity in atherosclerotic lesion was significantly increased after DE exposure, n=9, *P<0.02; Movat staining for foam cells E) (200X); F) (400X) G) DE exposure significantly increased foam cells formation, n=8, *P<0.04. Values are mean±SEM.  67  Volu me Fr action of α -actin Expression (V/v%) (Normalized toPlaque Area)  Volume Fraction o f Positiv e Staining for Oil Red O (V/v %) (No rmalized to Plaque Are a)  Filtered Air DE  A B  50µm  C  40  30  40  30  Filtered Air DE  D  Filtered Air DE  Filtered Air  *  20  10  0  Filtered Air DE  E  F  *  20  10  0  DE  68  Filtered Air  DE  G  H  100µm  Volume Fraction of Positive Staining for Picro-Sirius Red (V/v%) (Normalized to Plaque Area)  I 1.5  Filtered Air DE  1.0  0.5  0.0 Filtered Air  DE  Figure 3.4 Histochemical analysis of the components of atherosclerotic plaque in aortic root. A,B) Representative photomicrographs of Oil red O staining for lipid content (200X); C) Exposure to DE increased lipid content occupied in atherosclerotic lesions, n=6, *P<0.02; D,E) Staining of α-actin for smooth muscle cells (200X), insets are higher magnification (400X) photomicrographs showing smooth muscle cells in the plaque; F) The smooth muscle cell content was significantly increased after DE exposure, n=8, *P<0.05; G,H) Picro-sirius red staining for collagen content (100X); I) DE exposure induced a trend of reduction in collagen content, n=10, P=0.14. Values are mean±SEM.  69  3.3.5 Increased Oxidative Stress in Atherosclerotic Plaque The expression of tissue oxidative stress markers (iNOS, CD36, and Nitrotyrosine) in plaques were significantly increased after DE exposure; iNOS (18.7±5.5% vs. 50.8±12.2%; Filtered air vs. DE; p<0.05) (Fig.3.5A,B,C), CD36 (36.1±5.6% vs. 64.6±8.9%; Filtered air vs. DE; p<0.05) (Fig.3.5D,E,F), and nitrotyrosine (43.4±8.9% vs. 75.6±8.6%; Filtered air vs. DE; p<0.02) (Fig.3.5G,H,I). To further examine the relationship between the magnitude of DE exposure and the down stream effects, we examined the association between the number of alveolar macrophages positive for particles and the number of foam cells, the expression of CD36 and iNOS in plaques, respectively. As the number of alveolar macrophages positive for particles in filtered air group is significantly lower than that in DE exposure group (3.5±1.2% in F Filtered air vs.85.7±1.7% in DE), we used data from DE exposed mice. We found that there were positive correlations between alveolar macrophages positive for particles and plaque foam cell formation (R2=0.35, p=0.027), the CD36 expression (R2=0.49, p=0.015) and the iNOS expression (R2=0.72, p=0.0081), respectively (Fig.3.6).  70  Volume F raction of CD36 Expr ession (V/v %) (Normalized to Plaque Are a)  Vo lume F raction of iNOS Expre ssion (V/v%) (No rmalized to Plaque Are a)  Filtered Air DE  A B  100µm  80  60  80  60 Filtered Air DE  C  D  Filtered Air  *  40  20  0 Filtered Air DE  Filtered Air DE  E  F  Filtered Air DE  *  40  20  0  DE  71  Filtered Air  DE  G  H  50µm  Volume Fraction of NT Expression (V/v %) (Normalized to Plaque Are a)  I 100 80  Filtered Air DE  *  60 40 20 0 Filtered Air  DE  J  Figure 3.5 Immunohistochemical analysis of the expression of oxidative stress markers in the aortic root lesions. A,B) Representative photomicrographs of immunohistochemical staining for iNOS expression in aortic root (100X); C) Exposure to DE increased iNOS expression, n=8, *P<0.03; D,E) Immunohistochemical staining for CD36 expression (100X); F) CD36 expression was significantly increased after DE exposure, n=9, *P<0.05; G, H) Immunohistochemical staining of nitrotyrosine (NT) expression (200X); I) DE exposure significantly enhanced nitrotyrosine expression, n=9, *P<0.04; J) Negative control for H (200X). Insets are photomicrographs with higher magnification (400X). 72  80 60  B Volume Fraction of CD36 (V/v %) (Nor malized to Plaque Ar ea)  Volume Fraction of F oam Cells(V/v%) (Nor malized to Plaque Are a)  A y=0.7041x-35.469 R2 =0.35  40 20 0 60 -20  80  100  Alveolar Macrophages Positive for Particles (%)  150 y=1.832x-80.555  R2 =0.49 100 50 0 60  70  80  90  100  Alveolar Macrophages Positive for Particles (%)  -50  Volume Fraction of iNOS (V/v%) (Nor malized to Plaque Ar ea)  C 100 80  y=1.213x-49.098 R2 =0.72  60 40 20 0 50  60  70  80  90  100  Alveolar Macrophage s Positive for Particle s (%)  Figure 3.6 Association of DE exposure, plaque foam cell formation and the oxidative stress markers. A) Positive correlation between increased foam cell formation and alveolar macrophages positive for particles, P=0.027; B) Positive correlation between enhanced CD36 expression in aterosclerostic lesion in aortic root and alveolar macrophages positive for particles, P=0.015; C) Positive correlation between iNOS expression in aortic root and alveolar macrophages positive for particles, P=0.0081.  73  3.3.6 Systemic Oxidative Stress Urine levels of both 15-F2t-isoprostane (11.7±3.4 vs. 27.7±4.1ng/creatinine (mmol); Filtered air vs. DE; p<0.02), and 8-hydroxy-2-deoxy guanosine (1422±106 vs. 5110±1452 ng/creatinine (mmol); Filtered air vs. DE; p<0.02) were increased after DE exposure (Fig.3.7). The plasma MPO levels were similar between DE and filtered air exposure groups (Fig.3.8).  40  B  8-OH-dG(ng) / Cr eatinine (mmol)  8-isoprostane (ng) / Cre atinin e (mmol)  A  Flitered Air  *  DE 30 20 10 0 Flite red Air  8000  Filtered Air DE  4000 2000 0 Filtered Air  DE  *  6000  DE  Figure 3.7 Analysis of systemic oxidative stress. A) Increased protein oxidation supported by enhanced urine 8-isoprostane production, n=10, *P<0.02; B) Increased DNA oxidation demonstrated by augmented urine 8-OH-dG production, n=11, *P<0.02. Values are mean±SEM.  Relative Fluorescence  600  Filtered Air DE  400  200  0 Filtered Air  DE  Figure 3.8 Plasma myeloperoxidase (MPO) levels. The MPO levels were not significantly different between filtered air and DE exposed mice (n=10). Values are mean±SEM.  74  3.4 Discussion  In this study, we demonstrate that DE exposure induced changes in atherosclerostic plaque that are characteristic of unstable plaques associated with vascular events (294). We show that exposure to DE increased plaque lipid content, plaque cellularity, foam cell formation, and smooth muscle cell migration into plaques (Fig.3.5 & Fig.3.7). The positive correlation between increased plaque foam cell formation, and alveolar macrophages positive for particles (Fig. 3.6A), links DE exposure to the compositional changes in atherosclerotic lesions. We also show an increase in oxidative stress markers both in the circulation (e.g., 15-F2t-isoprostane and 8-OH-dG) and in the plaques themselves (e.g., iNOS, CD36, and NT) (Fig. 3.5 & Fig.3.7), and the positive association between particles in the lung and the increased expression of iNOS and CD36 in aortic root (Fig.3.6B,C) suggests that oxidative stress contributes to the DE-induced compositional changes. With the rapid industrialization in both developed and developing world, DE has become one of the most important components of urban particulate matter (15). This is the first study to quantify the morphological changes in atherosclerotic plaque induced by exposure to DE and linked these changes to oxidative stress. In this study, we exposed ApoE knockout mice to DE for 7 weeks using a wellcontrolled inhalation system that mimics real-world exposure near roadways. The average calculated exposure throughout a 24-hour period in our study was less than 35 µg/m3, which is within the range of the National Ambient Air Quality Standard for PM2.5 (22). We demonstrate that exposure to DE resulted in an increase in alveolar macrophages that phagocytosed particles, and the total number of alveolar macrophages (Fig.3.1), indicating the lung inflammation. Alveolar macrophages are key cells in processing inhaled particulate matter, and this study confirms previous findings from our laboratory that used a different exposure model (EHC93 instillation) (79; 287). Alveolar macrophages are also 75  powerful producers of the mediators (such as IL-1β, IL-6, IL-8, and GM-CSF) that fuel the systemic inflammatory response when processing inhaled ambient PM (79). These proinflammatory mediators are known to activate the endothelium and up-regulate VCAM-1, ICAM-1, selectins, which are the key steps for monocyte migration into the subendothelium to engorge lipid, hence promoting atherogenesis (140). We have recently shown that mediators produced in the lung translocate to the blood stream following PM10 exposure (107), underscoring the importance of the lung inflammatory response induced by PM10 exposure to the downstream vascular responses (1;10). These findings support the concept that the systemic inflammatory response is responsible for the downstream adverse vascular effects following exposure to ambient PM. The association between lung alveolar macrophages phagocytosed particles and foam cell formation in plaques (Fig.3.6A) supports the link between the magnitude of exposure to DE and atherogenesis.  3.4.1 Compositional Changes in Atherosclerotic Plaque Instability and rupture of an atherosclerotic plaque are implicated in the majority of acute ischemic syndromes, including MI and stroke. Histopathological features of these vulnerable plaques in humans include a large necrotic core (> 40% of plaque area), increased inflammatory cells (including macrophages/foam cells) with less smooth muscle cells, and a thin fibrous cap (<65 µm) accompanied by reduced collagen and proteoglycans. Although the features of vulnerable plaque in animal models are controversial (295), the features in murine atherosclerotic lesions, including a large lipid core, increased inflammatory cells (e.g., macrophages), can be considered as histological markers for plaque progression and vulnerability (294). Our study shows that exposure to DE promoted compositional changes in plaques, such as an increase in lipid accumulation, higher cellularity, more foam cell formation, and 76  smooth muscle cell content (Fig.3.3 & Fig.3.4). Studies from other (7; 8), and our own laboratories (6) suggest that exposure to ambient PM is associated with the development and progression of athereosclerosis. Using Watanabe heritable hyperlipidemic rabbits, and instillation of PM10 into the lung, we previously demonstrated that exposure to PM10 collected over a major North American city (EHC93) not only caused an increase in the extent of atherosclerosis (6), but also induced compositional changes in plaques (such as increase cellularity and lipid accumulation), similar to what we found in this study using an inhalation model. In subsequent studies using the rabbit model, we showed increased adhesion molecule expression in the endothelium overlying plaques and acceleration of monocyte migration into atherosclerotic lesions (133). Using a high fat diet mouse model, Sun and colleagues demonstrated that 6-month inhalation exposure to concentrated ambient PM2.5 particles enlarged plaque area and increased lipid content of atherosclerosis (8). Similar to the method of Sun’s study, we also used ApoE knockout mice and inhalation exposure, but we fed these mice with regular chow and our exposure period was shorter (7 weeks vs. 6 months). Albeit the shorter exposure period, we found more extensive compositional modifications of atherosclerotic lesions (Fig.3.3 & Fig.3.4), and enhanced oxidative stress in plaques (Fig.3.5), suggesting that DE may be more active in modifying atherosclerotic plaques (296), and particle composition may play an important role in PMinduced adverse cardiovascular effects (297). In the study, we did not observe significant differences of body weight, food consumption, and lipid levels (Table 3.1) between two groups. Using aortic root as a representative of large vessel atherosclerosis, we show that the volume fraction of the size of atherosclerotic lesions was not different between DE and filtered air exposed mice (Fig.3.3A,B). This results support findings from a recently published study using an even longer DE exposure model (297). Studies in humans showed that the majority of acute 77  vascular events originate in active plaques with more lipid accumulation and inflammatory cells (e.g., macrophages) (148), and this phenomena underlines the importance of plaque composition rather than plaque size as a risk for vascular events. We show that DE exposure caused increased foam cell formation, cells originate from circulating monocytes (133) that differentiate into macrophages that specialize in phagocytosis of modified LDLs via their scavenger receptors, such as CD36, thereby transforming into foam cells (140). Lipid uptake by macrophages stimulates the release of various cytokines, which support foam cell formation and smooth muscle cell recruitment from the media into the intima. Proinflammatory mediators (e.g., IL-1, IL-6, MIP-1α, and TNFα) promote smooth muscle cell migration, contributing to the evolvement of smooth muscle cells from “contractile" phenotype state to the active "synthetic" state. Synthetic smooth muscle cells synthesize proinflammatory cytokines, release matrix metalloproteinases that can digest the components of extracellular matrix, such as collagen and proteoglycan, and lead to weakening the integrity or the stability of plaques (298). Our findings of increased plaque lipid content, increased cellularity with more foam cells and smooth muscle cells support the notion that DE exposure causes a persistent inflammatory milieu in plaques, all hallmarks of unstable vulnerable plaques. As mentioned, human studies have shown that plaque activity reflected by plaque composition rather than plaque size is a stronger indicator for downstream vascular events (27). We postulate that DE-induced compositional changes in atherosclerotic plaque could explain at least part of the epidemiological associations between traffic pollution and cardiovascular events (257: 290) .  3.4.2 Oxidative Stress and Compositional Changes in Atherosclerotic Plaque Our results suggest that DE exposure induces oxidative stress that contributes to the compositional changes observed in atherosclerotic plaque. This notion is supported by the 78  increased expression of iNOS, CD36, and nitrotyrosine in atheroslclerotic lesions (Fig.3.5), enhanced systemic lipid and DNA oxidation, and the association between alveolar macrophages that phagocytosed particles (magnitude of exposure) and increased surrogate markers for tissue oxidative stress: iNOS and CD36 (Fig.3.6B,C). ROS generation can arise directly from the surface of ambient particles and soluble compounds (e.g., transition metals and/or organic compounds) (287). In addition, the airway alveolar macrophages are the first cells to come in contact with inhaled particles, and these cells release a large number of proinflammatory mediators upon DE stimulation, at the same time, they produce ROS at the sites of inflammation, resulting persistent local and systemic inflammatory response (287). Nitric oxide (NO) is an important biological mediator in living organisms that is synthesized from L-arginine using NADPH and molecular oxygen. The overproduction of NO catalyzed by iNOS is cytotoxic. iNOS is not expressed under physiology conditions, but up-regulated by pro-inflammatory mediators. iNOS not only catalyze NO production, but also generate superoxide in macrophages (299). The concomitant NO production and the accompanied increase in the level of superoxide lead to the generation of a strong NO-derived oxidant, peroxynitrite, which is implicated in the pathogenesis of a number of cardiovascular diseases, including atherosclerosis (217). Oxidative modification of LDL accelerates lipoprotein-uptake by macrophages promoting the formation of foam cells, which is the landmark of development of atherosclerosis. This oxLDL uptake is mainly mediated by a macrophage scavenger receptor CD36, also known as oxLDL receptor (288). CD36 is regulated by proatherogenic cytokines and lipids. We suspect that the enhanced CD36 expression in plaques promotes the increased foam cell formation, and lipid accumulation as we observed (Fig.3.3G & Fig.3.4C). Collectively, the increases in both systemic and tissue oxidative stress markers support our  79  hypothesis that exposure to DE induces ROS, which promote the compositional changes we observed in the atherosclerotic lesions.  3.5 Conclusions  In summary, we demonstrate that DE exposure results in specific changes in atherosclerotic plaque that are associated with plaque activity and vulnerability. We also show a strong association between the compositional changes in plaques and the particle uptake in alveolar macrophages. The association between traffic related air pollution exposure and vascular events is well established in epidemiology studies (22), and our study support the notion that changes in atherosclerotic plaques induced by traffic related pollutants, (e.g., DE), contribute to the vascular events.  80  Chapter 4 Exposure to Diesel Exhaust Up-regulates iNOS Activity in ApoE Knockout Mice1  4.1 Introduction  Numerous epidemiological studies have demonstrated an association between exposure to particulate matter air pollution with diameter less than 10µm (or called PM10) and increased cardiovascular morbidity and mortality (42; 56;57). Recent studies also showed that reducing PM10 levels resulted in a declined in cardiovascular deaths by 10.3% (37; 38), suggesting a causal effect of PM10 and cardiovascular mortality. It has been well established that deposition of ambient particles in the lung provokes low-grade alveolar inflammation with a secondary systemic inflammatory response resulting in downstream cardiovascular dysfunction. Atherosclerosis has been recognized as a chronic inflammatory disorder of blood vessels involving the vascular, metabolic, and immune systems(140; 285). We have previously shown that exposure to PM10 caused progression of atherosclerosis of coronary arteries and aortae in rabbits that naturally developed atherosclerosis (141). This sentinel finding was confirmed in ApoE knockout mice (143) and a human study by showing an association between progression of carotid atherosclerosis and levels of ambient air pollution --------------------------------------------------------------------------------------------------------------1  A modified version (with more detailed Methods) of this chapter has been accepted for publication by Toxicology and Applied Pharmacology. Ni Bai, Takashi Kido, Terrance J. Kavanagh, Joel D. Kaufman, Michael E. Rosenfeld, Cornelis van Breemen, Stephan F. van Eeden (2011) .  81  in Los Angeles (144). The progression of atherosclerosis induced by PM10 exposure could contribute to the increased cardiovascular morbidity and mortality. The observations of PM10-induced atherosclerosis progression are very compelling; however, the mechanisms whereby PM10 exposure causes progression in atherosclerosis, have not been fully elucidated. Diesel exhaust (DE) is a mixture of fine particles and gases, and represents a useful model of traffic-related air pollutants, which accounts for up to 90% of the fine particulate mass in ambient air of many major cities, such as London (15). A recent study demonstrated an association between progression of atherosclerosis and living near major roads (300). In addition, 6% of coronary heart disease deaths were linked to traffic-related pollution (15; 293). Inducible nitric oxide synthase (iNOS) is up-regulated in response to inflammatory cytokines as part of the host defense responses (286), and generates 100-1000-fold more nitric oxide (NO) than does endothelial NOS (eNOS) (micromolar vs. nanomolar levels). NO derived from eNOS plays an important role in protecting vasculature from inflammation and atherosclerosis. However, excessive NO production from iNOS has detrimental effects on cardiovascular function. The large amount of locally released NO has been linked to the generation of harmful oxidative products, such as peroxynitrite, which is implicated in iNOS-mediated development of atherosclerosis (143; 186; 189). iNOS is undetectable under normal physiological conditions, but its expression can be found in macrophages, endothelial cells and smooth muscle cells of human atherosclerotic plaques (214). The notion that iNOS plays a causative role in the progression of atherosclerosis is supported by the observation that atherosclerotic lesions were diminished in iNOS/ApoE double knockout mice, compared with ApoE knockout mice (218). iNOS overexpression was shown to be responsible for DEinduced lung inflammation (143; 186), while iNOS knockout mice had a significant reduction in cytokine levels in the lung after exposure to ambient PM (271). iNOS 82  expression is up-regulated by NF-κB, which is sensitive to inflammation and oxidative stress stimulation (301). We recently have shown that DE exposure accelerates the progression of atherosclerosis in ApoE mice via oxidative stress pathways. In this study, we test our hypothesis that DE exposure up-regulates iNOS expression in vasculature via a NF-κBmediated pathway that could ultimately contribute to the progression of atherosclerosis and cardiovascular events linked to exposure to traffic related pollution.  4.2 Methods and Materials (Please refer to Chapter 2 for details) 4.2.1 Methods 30-week old male ApoE knockout mice fed with regular chow were exposed to DE for 7 weeks (5days/week, 6hrs/day) at the concentration of 200 µg/m3. Exposing mice to filtered air was the control. Animal procedures were approved by the Animal Care and Use Committee of the University of Washington. After exposure, mice were euthanized with sodium pentobarbital (100mg/kg) and heparin sulfate (500U/kg) intraperitoneally. Plasma was obtained after the centrifugation of the blood and stored at -80°C until assay. Thoracic aorta, aortic root, and heart were carefully dissected from their connective tissues and kept in appropriate conditions until assay. To measurement vascular tone and iNOS activity, the thoracic aorta was carefully cleaned off connective tissues without damaging the endothelium, and mounted in a wire myograph. Smooth muscle contractility was studied by the addition of cumulative concentrations of phenylephrine (PE, 1nM-10µM). To examine the effect of DE on iNOS activity, a iNOS specific inhibitor (1400W) was used. iNOS activity was determined by the fractional changes of maximum PE constriction with and without the presence of 1400W.  83  Immunohistochemistry was performed to quantify the expression of iNOS, macrophages, smooth muscle cells, CD36, and nitrotyrosine in the thoracic aorta and aortic root sections that contained three complete valve leaflets by specific primary antibodies: iNOS (1:100); F4/80 (1:50); α-actin (1:600); CD36 (1:50); and nitrotyrosine (1:400). Western immunoblotting analysis was conducted to assess iNOS expression in heart tissue. Frozen heart tissues were homogenized and protein samples (40µg/lane) were separated on 7.5% SDS-PAGE gel, subsequently electrotransferred to polyvinyldifluoride membranes and blocked with 5% BSA dissolved in 0.1% TBS-Tween 20 for 60 min at room temperature. The membrane was incubated with specific primary antibody for iNOS (1:500) at 4°C overnight. Densitometry was carried out using Image J (NIH), and the results were expressed in optical intensity per mg protein. NF-κB (p65) activity was assessed on nuclear extracts from frozen heart tissue by enzyme-linked immunosorbent assay (ELISA) using a NF-κB (p65) transcription factor kit. Results were expressed as absorbance (450nm) per mg protein. Real-time reverse transcription polymerase chain reaction (RT-PCR) was conducted to quantify mRNA expression for iNOS and NF-κB. Total RNA was extracted from heart tissue and the same amount of RNA was loaded in triplicates for each assay, and qRT-PCR was performed using TaqMan universal PCR master mix and Taqman gene expression assay system (Applied Biosystems). mRNA expression for iNOS, NF-κB, β-actin, and hypoxanthine phosphoribosyltransferase-1 (HPRT1) was determined by qRT-PCR using ABI Prism 7900HT sequence detection system. Gene expression values were calculated and displayed as ratio relative to β-actin.  84  4.2.2 Solutions and Chemicals The PSS consisted of the following (in mM): NaCl 119, KCl 4.7, KH2PO4 1.18, NaHCO3 24, MgSO47H2O 1.17, CaCl2 1.6, glucose 5.5 and EDTA 0.026. All reagents were purchased from Sigma (St. Louis, MO). 4.2.3 Statistical Analysis Data are shown as mean±SEM. The statistical significance was evaluated using the unpaired Student t test for simple comparison between 2 values. Non-parametric analysis (Kruskall-Wallis test) was used to analyze data that were not normally distributed and Bonferroni correction was used for multiple comparisons. Linear regression modeling was used to assess the correlation relationship between parameters. In all experiments, n equals the number of mice from which samples were obtained. P<0.05 was considered to be significant.  4.3 Results  4.3.1 iNOS Activity and Expression in the Thoracic Aorta  PE-stimulated vasoconstriction of ApoE mouse thoracic aorta was suppressed by ~23% after DE exposure (9.5±0.6mN vs. 7.4±0.6mN; Filtered air vs. DE; p<0.02) (Fig.4.1A,B). In the presence of 1400W, the fractional changes of PE-elicited vasoconstriction were significantly increased in DE exposure group, compared with the control (5.2±3.0% vs. 24.1±7.6% of increased maximum constriction; Filtered air vs. DE; p<0.04) (Fig.4.1C,D,E), suggesting an increase in iNOS activity after DE exposure. To verify whether the reduced vasoconstriction was due to morphological abnormalities of blood vessels, we stained the thoracic aorta that was adjacent to the segment used for functional studies, with hematoxylin and eosin, and measured the thickness of the vascular 85  wall using quantitative morphologic analysis. We found no difference of the thickness and structure of the vessels after DE exposure, compared with the control (Appendix B). Immunohistochemical staining demonstrates that iNOS was predominantly expressed by smooth muscle cells and macrophages in the vessels (Fig.4.2A,B,C). The increased iNOS activity is supported by quantitative immunohistological analysis showing that the iNOS expression in thoracic aorta was significantly elevated (~4 fold) after DE exposure (11.9±3.2% vs. 40.2±6.5%; Filtered air vs. DE; p<0.002) (Fig.4.2D).  86  A  B  12  Tension (mN)  *  ** 15  Tension (max) (mN)  *  9  *  6  3  Filtered Air DEP  0 -9  -8  -7  -6  Filtered Air DEP  *  10  5  0  -5  Filtered Air  DEP  Log[PE](M)  C  D 12  Tension (mN)  Tension (mN)  12  8  4  8  4 DE  Filtered Air PE+1400W 0 -9  PE+1400W 0  -8  -7  -6  -5  -9  -8  -7  -6  -5  Log[PE](M)  Log[PE](M)  Fractional Changes of Tension (%)  E 40  Filtered Air 30  *  DE  20  10  0  50 µM PE+ 1400W  Figure 4.1 Reduced vasoconstriction and increased iNOS activity in the thoracic aorta. A) Dose-response curves of PE-elicited constriction show that exposure to DE attenuated vasoconstriction, n=9, *P<0.02; B) DE exposure caused significant attenuation of maximum vasoconstriction, compared with the control, n=9, *P<0.02; C) In the control group, the presence of iNOS blocker (1400W) had no effect on PEelicited constriction; D) In DE exposure group, the reduced vasoconstriction was partly restored by 1400W; E) Inhibition of iNOS by 1400W caused a significant elevation of maximum vasoconstriction in DE exposure group, n=9, *P<0.04. Values are mean±SEM. 87  A  B  C  10µm  D  Volume Fraction of iNOS Expr ession (V/v %) (Normalize d to Vascular Wall Th ickness)  50 40  Filtered Air DE  *  30 20 10 0 Filtered Air  DE  Figure 4.2 Immunohistochemical analysis of iNOS expression in the thoracic aorta. Representative photomicrographs of staining for A) iNOS (arrow); B) Macrophages (F4/80) (arrow); C) Smooth muscle cells (α-actin) (arrow); D) Exposure to DE increased iNOS expression in the thoracic aorta, n=8, *P<0.002. Values are means ± SE; L: lumen. Magnification: orginalX400. Values are mean±SEM.  88  4.3.2 iNOS Expression in the Heart Quantitative immunohistochemical staining shows that iNOS expression was increased in the heart tissues after DE expousure (73.5±10.0% vs. 116.9±7.6%; Filtered air vs. DE exposure; p<0.01) (Fig.4.3C). Consistently, western immunoblotting shows that DE exposure caused augmented iNOS expression in the heart tissues (0.7±0.1% vs. 1.0±0.1%; Filtered air vs. DE; p<0.03) (Fig.4.3D,E).  89  A  B  40 50µm  Volume Fraction of iNOS Expression (V/v %) (Nor malized to Car diac M uscle Ar ea)  C  150  Filtered Air DE  *  100  50  0 Filtered Air  DE  Filtered Air  iNOS  β-actin  E DE  130 KDa  40 KDa  iNOS Expression (%) (Ratio of Inte nsity of iNOS / β -actin)  D 1.5  Filtered Air DE  *  1.0  0.5  0.0 Filtered Air  DE  Figure 4.3 Immunohistochemical and Western blotting analysis of iNOS expression in the heart. Representative photomicrograph of staining for A) negative control; and B) iNOS in cardiac muscle cells; C) Quantitative immunostaining analysis shows that exposure to DE increased iNOS expression in the heart, n=9, *P<0.01; D) Representative photomicrograph of western blotting of iNOS expression in the heart; E) Quantitative western blotting analysis demonstrats that iNOS expression was enhanced following DE exposure, n=8, *P<0.03. Magnification: orginal X200. Values are mean±SEM. 90  4.3.3 Co-localization of iNOS, CD36 and Nitrotyrosine Expression iNOS was predominantly expressed in macrophages in the aortic root tissues (Fig.4.4B,C). The expressions of iNOS, CD36, and nitrotyrosine were co-localized in macrophages (Fig.4.4D,E,F), In addition, iNOS expression was positively associated with CD36 expression (R2= 0.458, P=0.01) (Fig.4.4G), and CD36 expression was correlated with nitrotyrosine formation (R2= 0.4629, P=0.01) (Fig.4.4H).  A  20µm  B  C  91  D  F  E  H  y=06783+32.9 R2 =0.458  Root NT Expression (V/v%)  Root CD36 Expre sssion (V/v%)  G 150  100  50  0 0  20 40 60 80 Root iNOS Expression (V/v%)  100  y=0.8026 x +22.68 R2 =0.4629  150  100  50  0 0  20 40 60 80 Root CD36 Expression (V/v%)  100  Figure 4.4 iNOS expression in atherosclerotic plaque in aortic root and its relationship with the expression of CD36 and nitrotyrosine. Represetitive photomicrographs of staining for A) Negative control; B) iNOS; and C) Macrophages (F4/80); co-localized expression of D) iNOS; E) CD36; and F) nitrotyrosine in aortic root; G) Positive correlation between iNOS and CD36, P=0.01; H) Positive correlation between CD36 and NT, P=0.01. Magnification: original X400.  92  4.3.4 NF-κB (p65)-Mediated iNOS Expression mRNA expression of iNOS (76±3% vs 90± 6%; filtered air vs. DE; p<0.004) (Fig.4.5A) and NF-κB (83±1% vs 88 ± 3%; Filtered air vs. DE; p<0.01) (Fig.4.5B) were significantly higher in the heart tissues of DE exposed mice than the control. To assess whether iNOS was regulated at the transcription level via NF-κB activation, we extracted nuclear protein from the frozen hearts, and NF-κB activity was evaluated by ELISA. DE exposure up-regulated NF-κB (p65) activity (0.014±0.0004 vs. 0.029 ±0.006 absorbance of 450nm/mg protein; Filtered air vs. DE; p<0.01) (Fig.4.6A). iNOS expression was positively correlated with NF-κB activation (R2= 0.5998, p<0.0005) (Fig.4.6B), suggesting that DE exposure up-regulated iNOS expression at transcriptional level via NF-κB activation (p65).  Filtered Air DE  *  80 60 40 20 0  Filtered Air  DE  100  F iltered Air DE  *  80 to β -actin (%)  100  B Relativ e NFκ κ B mRNA Expression  Re lative iNOS mRNA Expre ssion to β -actin (%)  A  60 40 20 0  Filtered Air  DE  Figure 4.5 Increased mRNA expression of iNOS and NF-κ κB in the heart. A) Increased mRNA expression for iNOS in the heart after DE exposure, n=7 (filtered air), n=8 (DE) *P<0.004; B) Exposure to DE increased NF-κB mRNA expression, n=7 (filtered air), n=8 (DE), *P<0.01. Values are mean±SEM.  93  B  0.04  Filtered Air DE  *  0.03 0.02 0.01 0.00 Filtered Air  DE  Volume Fraction of iNOS Expression (%) (Nor malized to Plaque Ar ea)  Absor bance (450nm) pe r mg pr otein  A  100 80  y = 3380-24.585 R2 =0.5998  60 40 20 0 0.010  0.015  0.020 0.025 0.030 NFκ κB Activity (absorbance (450nm) per mg protein)  Figure 4.6 Increased NF-κ κB activity of heart nuclear extract. A) The NFκB activity in the heart was significantly increased after DE exposure (n=10, *P<0.01). Values are mean±SEM; B) The increased NF-κB activity was positively correlated with iNOS expression.  4.4 Discussion  Studies from our (141) and other (143; 144) laboratories have shown that exposure to PM10 is associated with development of athereosclerosis. In this study, we show that exposure to DE, a major contributor to urban particulate matter, increased iNOS expression in the thoracic aorta and heart tissues. iNOS was expressed by smooth muscle cells, macrophages, and cardiac myocytes (Fig.4.2, Fig.4.3B, Fig.4.4B). DE exposure enhanced iNOS activity in blood vessels, which resulted in decreased vascular contractility (Fig.4.1). We have recently reported DE exposure enhanced iNOS expression in plaque tissues of the aortic root (377). In this study, we show that the expression of iNOS, CD36 and nitrotyrosine were all co-localized in the plaque tissues (Fig. 4.4D,E,F). There was a positive association between enhanced NF-κB activity and iNOS expression (Fig.4.6B), suggesting that the activation of NF-κB plays a pivotal role in the enhanced iNOS activity. The augmented iNOS and NF-κB mRNA expression (Fig.4.6) also indicate that iNOS was up94  regulated at both transcription and translation levels. To our knowledge, this is first study demonstrating that DE exposure up-regulates iNOS activity and expression in the vasculature as well as the heart tissues. We speculate that oxidative stress-induced NF-κB activation contributes to the increased iNOS expression and activity.  4.4.1 The Level of DE Exposure In the study, we exposed ApoE knockout mice to DE for 7 weeks, using a wellcontrolled inhalation system that mimics real-world exposure. Our average particulate matter concentration throughout a 24-hour period is less than 35 µg/m3, which is environmentally relevant and within the National Ambient Air Quality Standard (8).  4.4.2 DE Exposure and Up-regulation of iNOS Nitric oxide (NO) is an essential biological mediator protecting the vessels from inflammation and atherosclerosis. Nevertheless, overproduction of NO catalyzed by iNOS is cytotoxic. iNOS is not expressed under normal physiological conditions, but can be upregulated in response to pro-inflammatory mediators, and oxidative stress stimulation. Upregulation of iNOS is implicated in the pathogenesis of a number of cardiovascular diseases, including atherosclerosis (217). In this study, we showed that DE exposure increased iNOS activity (Fig.4.1) and enhanced iNOS expression in the blood vessels (Fig.4.2) and heart (Fig.4.4) at both protein and mRNA levels (Fig.4.5A). We also found a positive correlation between the iNOS expression with both foam cell formation and the number of smooth muscle cells in atherosclerotic plaque in aortic root (Appendix C). The association suggests that the up-regulation of iNOS contributes to the progression of atherosclerosis induced by DE exposure. Similar to the finding of Sun and colleagues, in which ApoE knockout mice were exposed to concentrated ambient particles for 6 months (143), we found that iNOS 95  expression was up-regulated at both protein and mRNA levels after exposure to DE for a shorter exposure period (7 weeks vs. 6 months), suggesting that the constituents of DE may be more active, comparing with concentrated ambient particles (296). Sun and colleagues found that exposure to concentrated ambient particles exaggerated vasoconstriction to phenylephrine, which likely resulted from reduced NO production or endothelial dysfunction. However, exposure to DE for 7 weeks appeared to have a greater effect on smooth muscle cells, and we found suppressed vasoconstriction to phenylephrine, which was likely due to excessive NO production derived from iNOS (Fig 4.1). In addition, no impairment of AChstimulated endothelium-dependent relaxation was observed in our study (Fig 6.1A). Sun and colleagues were also observed that mice fed with regular chow did not develop endothelial dysfunction after exposure to concentrated ambient particles (supporting our finding), suggesting that high fat diet-mediated modifications on vasculature are important elements for ambient particle exposure-induced endothelial dysfunction. iNOS activation is mainly regulated at transcriptional level by NF-κB (302). Oxidative stress-induced NF-κB activation has been observed in epithelial cells exposed to ambient particles (105). NF-κB is known to regulate the transcription of various genes participating in oxidative stress responses. NF-κB dimers (p50/65) are sequestered in the cytoplasm by a family of inhibitors, called inhibitors of κB (IκB). Activation of NF-κB is initiated by degradation of IκB proteins triggered by oxidative stress. NF-κB complex enters into the nucleus in which it can turn on the expression of specific genes by binding to their promoters. In this study we found that NF-κB activity and mRNA expression were increased (Fig.4.5B & Fig.4.6A). The correlation between iNOS and increased NF-κB activity (Fig.4.6B) suggests that NF-κB contributes to the up-regulation of iNOS following DE exposure. The promoter of the murine gene coding for iNOS contains the binding sites for NF-κB, which are sensitive to oxidative stress stimulation (303). DE exposure was found to cause ROS 96  generation in epithelial cells, alveolar macrophages, blood vessels, endothelial cells, and cardiac myocytes (105;177; 180; 186; 304). We recently demonstrated that DE exposure increased oxidative stress in atherosclerotic lesions in the aortic root, and systemic protein and DNA oxidation (377). In this study, we showed that the enhanced CD36 and nitrotyrosine were concomitantly up-regulated with iNOS (Fig.4.4), suggesting that ROS may play a role in the activation of NF-κB, which mediated the up-regulation of iNOS expression and activation. ROS generation can rise directly from the surface of ambient particles (305), and soluble compounds (e.g., transition metals, or organic compounds (183; 296; 308). In addition, direct interactions between DE and airway epithelial cells and alveolar macrophages could also contribute to ROS generation (287; 309).  4.4.3 iNOS and Atherosclerosis The notion that overexpression of iNOS results in development of atherosclerosis has previously been established by showing that atherosclerotic lesions were attenuated when iNOS was knocked out in ApoE mice (218; 269). iNOS is capable of generating superoxide, which can rapidly deplete endothelium-derived NO, attenuate eNOS activity and expression, leading to endothelial dysfunction and development of atherosclerosis. It is well known that oxidative modification of low-density lipoprotein (LDL) accelerates lipoprotein-uptake by macrophages and the formation of foam cells (310), which is the characteristic component of atherosclerotic lesions and landmark of development of atherosclerosis. This oxidized LDL (oxLDL) uptake is mainly mediated by a macrophage scavenger receptor CD36, also known as oxLDL receptor (311). We previously reported that DE exposure increased CD36 expression. CD36 is up-regulated by proatherogenic cytokines and oxidized lipid. In this study we showed that the expression of iNOS and CD36 was co-localized in atherosclerotic lesions (Fig.4.4D,E). The positive correlation between iNOS and CD36 (Fig.4.4G) suggests 97  that iNOS, as a free radical generator, may also play a role in the augmented CD36 expression, which contributes to the foam formation and progression of atherosclerosis. In addition to generating free radicals, iNOS also contributes to the production of peroxynitrite, which is a potent oxidant and implicated in the pathogenesis of several cardiovascular diseases, including atherosclerosis, MI, and heart failure (312).In summary, we show that DE exposure enhanced iNOS activity and caused up-regulation of iNOS at both protein and mRNA levels. We also show that DE induced oxidative stress, and increased NF-kB expression and activity, suggesting a pathway of the up-regulation of iNOS expression and activity. Our study provides novel insights into the mechanisms underlying DE exposureinduced progression of atherosclerosis, and potential future therapeutic targets to decrease the impact of ambient PM on cardiovascular morbidity and mortality.  98  Chapter 5 Exposure to Diesel Exhaust Causes Up-regulation of COX2 Activity in ApoE Knockout Mice  5.1 Introduction  Epidemiological studies have shown that exposure to ambient particulate matter air pollution with diameter less than 10µm (PM10) is an independent risk factor for cardiovascular morbidity and mortality (3; 22; 287). The mechanisms underlying PM10 induced cardiovascular disease have not been fully investigated. Both human and animal studies have indicated that the deposition of particles in the lung provokes a low-grade lung inflammation with a secondary systemic inflammatory response charactierized by increased circulating pro-inflammatroy mediators, such as IL-1, IL-6, and TNFα. The systemic responses are thought to cause the downstream cardiovascular diseases (22; 287). Prostanoids play an important role in cardiovascular function, such as regulating vascular tone, modulating vascular inflammatory response, controlling leukocyte-endothelial cell adhesion and platelet aggregation (368). Cyclooxygenase (COX) is the key regulatory enzyme responsible for the formation of prostanoids. COX catalyzes the conversion of arachidonic acid to prostaglandin (PG) H2, which is subsequently converted to a variety of eicosanoids, including PGE2, PGD2, PGF2α, prostacyclin I2 (PGI2), and thromboxane A2 (TXA2) (245). The array of PGs produced varies, depending on the downstream enzymes present in different types of cells. For example, endothelial cells primarily produce PGI2, whereas platelets mainly produce TXA2. Two COX-isozymes, COX1 and COX2, have been 99  characterized. COX1 is the enzyme responsible for basal, constitutive prostaglandin synthesis, whereas COX2, like iNOS is implicated in various inflammatory settings (313). PGI2 (vasodilator) and TXA2 (vasoconstrictor) are important components of prostaglandin metabolism that regulate cardiovascular homeostasis. Under physiological conditions, PGI2 is released by endothelial cells to mediate several protective effects on the vascular wall, including regulation of vascular tone, and inhibition of platelet aggregation and adhesion (314; 315). TXA2, a potent vasoconstrictor, is the predominant mediator opposite to the effect of PGI2. Under pathological conditions, such as inflammation or atherosclerosis, PGI2 production decreases, and the release of TXA2 becomes dominant (316). TXA2 is implicated in endothelial dysfunction, hypertension, and atherothrombosis. Exposure to PM10 causes overexpression of COX2 in human airway epithelial cells, resulting in the lung inflammation and injury (274). In vitro study showed that exposure to diesel exhaust particels increased COX2 protein and mRNA expression that were associated with a subsequent increase in cholesterol accumulation and foam cell formation (275). We previously showed that exposure to PM10 caused progression of atherosclerosis in rabbits (141), and recently demonstrated that exposure of ApoE knockout mice to diesel exhaust (DE) for 7 weeks promoted compositional changes of atherosclerotic lesions (e.g., increased foam cell formation, and lipid accumulation) (377). The underlying mechanisms for the changes in plaques are not fully understood. We hypothesize that DE exposure up-regulates COX2 activity and expression that could contribute to the DE exposure-induced compositional changes in atherosclerotic plaque.  100  5.2 Methods and Materials (Please refer to Chapter 2 for details) 5.2.1 Methods Male ApoE knockout mice at the age of 30 weeks, were exposed to DE for 7 weeks (5days/week, 6hrs/day) at the concentration of 200 µg/m3. Exposing mice to filtered air was the control. Animal procedures were approved by the Animal Care and Use Committee of the University of Washington. Plasma was obtained after the centrifugation of the blood and stored at –80°C until assay. The thoracic aorta, aortic root, heart, and lung were carefully dissected from their connective tissue and kept in appropriate solution until assay. The thoracic aorta was cut to 2mm rings and mounted on a wire myograph. Smooth muscle contractility was studied by the addition of cumulative concentrations of phenylephrine (PE, 1nM-10µM). To access the effect of DE exposure on COX, indomechacin (1µM) was applied. To examine the specific roles of COX1 and COX2, SC560 and NS398 were used to inhibit COX1 and COX2, respectively. Aortic root sections were incubated with specific primary antibodies: rabbit anti mouse antibody for COX1 (1:150), rabbit anti mouse antibody for COX2 (1:100), and rat anti mouse antibody for F4/80 (1:50) at 4°C overnight. The positive red staining was recognized, quantified by colour segmentation (Image Pro Plus), and normalized to the area of the atherosclerotic lesion. PGI2 level was assessed by measuring urine 2,3-dinor-6-keto prostaglandin F1α, the major urinary metabolite converted from PGI2, using an EIA kit. Creatinine levels were measured using a creatinine assay kit. Due to the limited quantity of vessel samples, total RNA was extracted from the heart tissues using RNeasy Fibrous Tissue Mini Kit (Qiagen). The mRNA expression of COX1, COX2, PGI2, β-actin, and hypoxanthine phosphoribosyltransferase-1 (HPRT1) was 101  measured by qRT-PCR (TaqMan) using the ABI Prism 7900HT sequence detection system (Applied Biosystems).  5.2.2 Solutions and Chemicals The PSS consisted of the following (in mM): NaCl 119, KCl 4.7, KH2PO4 1.18, NaHCO3 24, MgSO47H2O 1.17, CaCl2 1.6, glucose 5.5 and EDTA 0.026. All reagents were purchased from Sigma (St. Louis, MO).  5.2.3 Statistical Analysis  Results are reported as mean±SEM. The statistical significance was evaluated using the unpaired Student’s t test for simple comparison between two values. The concentrationresponse curves of the different groups were compared by ANOVA for repeated measurements followed by Bonferroni’s correction. P<0.05 was considered to be significant. In all experiments, n equals the number of mice from which samples were obtained.  5.3 Results  5.3.1 Vascular Constriction and COX Activity We found that PE-induced constriction was significantly suppressed in DE exposure group, compared with the control group (Fig.5.1). In the presence of indomechacin, PEelicited constriction was reduced in both control and DE exposure groups (Fig.5.2A,B). In addition, the fractional changes of attenuated constriction was significantly reduced in DE group (31.7±4.6% vs 12.6± 4.2%, Filtered air vs. DE, p<0.02) (Fig.5.2C), suggesting a modification of COX activity after DE exposure, and an imbalance of COX-mediated metabolites towards to an increased production of relaxant.  102  12  Tension (mN)  * 8  *  **  *  4  Filtered Air DE 0 -9  -8  -7  -6  -5  Log[PE](M)  Figure 5.1 Reduced vasoconstriction in the thoracic aorta. Exposure to DE attenuated PE-stimulated vasoconstriction, n=9, *P<0.05. Values are mean±SEM.  103  A *  8  12  ∗*  *  Tension (mN)  Tension (mN)  12  * *  4  B  8  4  PE (Filtered Air) PE+Indomethacin  PE (DE)  PE+Indomethacin  0  -9  -8  -7  -6  0  -5  -9  -8  Log[PE](M)  -7  -6  -5  Log[PE] (M)  C  Filtered Air DE  Fractional changes of tension of PEmax (%)  40 30 20  *  10 0 Filtered Air  DE  50 µM PE+ Indomethacin  Figure 5.2 Reduced vasoconstriction in the presence of COX inhibitor (indomethacin) and reduced fractional constriction in the thoracic aorta. Concentration-response curves of PE-stimulated vasoconstriction shows that COX inhibitor caused significant reduction of constriction only in filtered air exposed mice (A) (n=9, *P<0.001), but not in DE group (B), n=9, p=0.17; C) The fractional changes of maximum vasoconstriction in the presence of indomethacin were significantly lower in DE exposure group than the control, n=9, *P<0.02. Values are mean±SEM.  104  5.3.2 Immunohistochemical Staining Analysis of COX1 and COX2 Expression in the Thoracic Aorta and Aortic Root In the thoracic aorta, COX1 was expressed by endothelial cells, smooth muscle cells, and macrophages (Fig.5.3A,B,C,G)., COX2 was expressed by smooth muscle cells and macrophages (Fig.5.3D,E,F,G). In aortic root, COX1 and COX2 were expressed by macrophages (Fig.5.5B,C,D). The COX1 expression in the thoracic aorta and aortic root was similar between filtered air and DE exposure groups (Fig.5.4A,B,C & Fig.5.6A,B,C). We observed a significant enhancement of COX2 expression both in the thoracic aorta (1.1±0.1% vs 1.6 ± 0.1%, Filtered air vs. DE; p<0.007) (Fig.5.4D,E,F) and in aortic root (0.81±0.06% vs 0.99 ± 0.17%, Filtered air vs. DE; p<0.02) (Fig.5.6D,E,F). A  L  20µm  B  C  L  L  20µm  105  E  D  F4/8  50µm  F  G  L  L  20µm  H  I  L  L  Figure 5.3 Representative photomicrographs of staining in the thoracic aorta for A) COX1 (400X); B) COX1 (600X); C) Endothelial cells (von Willerband factor; 600X); D) COX2 (100X); E) Smooth muscle cells (α-actin; 100X); F) COX2 (400X); and G) Macrophages (F4/80; 400X); H) Negative control for A (400X); I) Negative control for F and G (400X); L: lumen.  106  Filtered Air  DE  A  B  L  L  Volume Fraction of COX1 Expre ssion (Vv%) (Normalized to Vascular Wall Thickness)  20µm  C 1.5  Filtered Air DEP  1.0  0.5  0.0 Filtered Air  DEP  Filtered Air  DE  D  E  L  Volume Fraction o f COX2 Expre ssion (Vv%) (No rmalized to Vascular Wall Thickness)  L  F 2.0  Filtered Air DEP  *  1.5 1.0 0.5 0.0 Filtered Air  DEP  Figure 5.4 Immunohistochemical analysis of COX1 and COX2 expression in the thoracic aorta. Representative photomicrographs of staining for COX1 (A,B) and COX2 (D,E) in the thoracic aorta of mice exposed to filtered or DE; C) COX1 expression was not altered after DE exposure, n=8, p=.02; F) COX2 expression was increased in DE exposure group, compared with the control, n=8, *p<0.007. L: lumen. Magnification: orginal X400. Values are mean±SEM. 107  A  B  20µm  100µm  C  D  F  20µm  Figure 5.5 Representative photomicrographs of staining in aortic root for A) Movat staining (100X); B) Macrophages (F4/80; 600X); C) COX1 (600X); and D) COX2 (600X); F) Negative control (400X).  108  Filtered Air  DE  A  50µm  B  Volume Fraction o f COX1 Exp re ssion (V/v%) (No rmalized to Plaque Are a)  C 1.5  Filtered Air DE  1.0  0.5  0.0 Filtered Air  DE  Filtered Air  DE  D  E  Volume Fraction of COX2 Expre ssion (V/v%) (No rmalized to Plaque Are a)  F 1.5  Filtered Air DE  *  1.0  0.5  0.0 Filtered Air  DE  Figure 5.6 Immunohistochemical analysis of COX1 and COX2 expression in aortic root. Representative photomicrographs of staining for COX1(A,B) and COX2 (D,E) in artic root; C) COX1 expression was not altered after DE exposure, n=8, p=0.3; F) COX2 expression was increased in DE exposure group, n=8, *p<0.02. Magnification: orginal X200. Inset in A is the negative control. Values are mean±SEM. 109  5.3.3 Urine 2,3-dinor-6-keto PGF1α Production The urinary excretion of 2,3-dinor-6-keto PGF1α is the major urinary metabolite of renal PGI2. The 2,3-dinor-6-keto PGF1α concentration was significantly increased by more than 15-fold after DE exposure, compared with the control (22.68±5.5 vs 344.6 ±  2,3-dinor-6-keto PGF1α (pg/ml) /Cr (mg/dl)  117.1pg/ml per Cr (mg/dl), Filtered air vs. DEP, p<0.007) (Fig.5.7).  500 400  Filtered Air DE  *  300 200 100 0  Filtered Air  DE  Figure 5.7 Urine 2,3-dinor-6-keto PGF1αα concentration. Urine 2,3-dinor-6-keto PGF1α concentration was significantly higher after exposure to DE than the control, n=10, *P<0.007. Values are mean±SEM.  5.3.4 Real-Time RT- PCR of the mRNA expression of COX1, COX2 and PGI2 in the Heart To examine whether the mRNA expression of COX1, COX2 and PGI2 was also altered, we extracted RNA from the heart tissue and performed quantitative real-time PCR using Taqman assay. We found no change in COX1 mRNA expression after exposure to DE, compared with the control (Fig.5.8A). However, the mRNA expression of COX2 was significantly increased after exposure to DE (38.0±1.8% vs 50.4 ± 3.6%, Filtered air vs. DE; p<0.006) (Fig.5.8B). The PGI2 expression in the heart was also significantly augmented after DE exposure, compared with the control (71.4±1.1% vs 78.4± 2.5%, Filtered air vs. DE; p<0.02) (Fig.5.8C).  110  B Relative COX2 mRNA Expression to β -actin (%)  Relative COX1 mRNA Expression to β -actin (%)  A Filtered Air DE  100 80 60 40 20 0  Filtered Air  DE  60  Filtered Air DE  *  40  20  0  Filtered Air  DE  Relative PGI2 mRNA Expression to β -actin (%)  C Filtered Air DE  100  *  80 60 40 20 0  Filtered Air  DE  Figure 5.8 The mRNA expression of COX1, COX2 and PGI2 in the heart. A) The mRNA expression of COX1 was not altered after exposure to DE, n=7 (filtered air), n=8 (DE), P>0.05; B) DE exposure significantly increased COX2 mRNA expression, n=7 (filtered air), n=8 (DE), *P< 0.006; C) The mRNA expression of PGI2 significantly increased in DE exposure group (n=8), compared with filtered air exposure (n=7), *P< 0.02. Values are mean±SEM.  5.4 Discussion  COX1 or/and COX2 play an important role in the progression of atherosclerosis (248; 273). Inhibition of COX1/COX2 has been associated with the reduction of cardiovascular events. Here, we explored the contribution of both COX1 and COX2 to the vascular dysfunction induced by exposure to DE.  111  In this study, we demonstrate that DE exposure caused up-regulation of COX2 activity and expression in blood vessels. We also show that DE exposure caused an increase in PGI2 production. We postulate that COX2 contributes to the excessive PGI2 production. In addition, DE exposure caused significant attenuation of PE-stimulated vasoconstriction. Prostanoid biosynthesis is significantly increased in response to inflammatory stimuli. Non-selective COX1/COX2 inhibitors, or non-steroidal anti-inflammatory drugs (NSAIDs), are widely used as anti-inflammatory agents, highlighting the pro-inflammatory role of the prostanoids. Traditionally, COX1 is recognized as a housekeeping enzyme that is constitutively expressed in endothelial cells, and vascular smooth muscle cells, and responsible for the production of physiological prostanoids to maintain cardiovascular homeostasis. Nevertheless, animal studies have shown that overexpression of COX1 is also implicated in a number of cardiovascular diseases, including hypertension (317) and atherosclerosis (273). The deleterious effects are mainly resulted from COX1-derived TXA2 production, a potent vasoconstrictor and platelet activator. TXA2 causes endothelial dysfunction and promotes vascular smooth muscle cell adhesion, migration, and proliferation (318). In this study, we examined COX1 protein and mRNA expression in the blood vessel, and aortic root. Consistent with previous reports, we found that COX1 was expressed by endothelial cells, smooth muscle cells, and macrophages (Fig.5.3). Our data show that DE exposure did not alter COX1 expression following DE exposure. In addition, we used selective TXA2 receptor antagonist (SQ29548), but failed to observe any modifications of vasoconstriction after DE exposure (Appendix D), suggesting that DE exposure did not have effects on TXA2 production that is predominantly regulated by COX1. Our results support the findings from other studies showing that the up-regulation of COX1 expression mainly occurs at the early, or acute stages of cardiovascular dysfunction (319).  112  COX2 is usually not detectable under normal physiological conditions, but can be upregulated by inflammatory or other pathological stimuli. Both human and animal studies have demonstrated that COX2 is implicated in the progression of atheroslcerosis (320; 321). DE exposure can up-regulate COX2 protein and mRNA expression in human macrophages (322). Exposure to residual oil fly ash induced overexpression of COX2 in the lung, whereby leading to acute lung injury (274). In addition, exposure to motorcycle exhaust particles caused up-regulation of COX2 in rat vascular smooth muscle cells and promoted smooth muscle cell proliferation (323). We found that COX2 was predominantly expressed in smooth muscle cells and macrophages in the vascular wall and aortic root (Fig.5.3, Fig 5.5). We show that DE exposure up-regulated COX2 expression at both protein and mRNA levels (Fig.5.4F, Fig.5.6F, Fig.5.8B). Vascular function study shows that PE-elicited constriction was attenuated (Fig.5.1), and we postulate that this was due to an imbalance between PGI2 and TXA2 production. This idea is supported by an excessive production of the major metabolite of PGI2 in the urine (Fig.5.7). In addition, PGI2 mRNA expression in the heart was also enhanced after DE exposure (Fig. 5.8C). PGI2 is mainly derived from COX2. Immunohistochemical and qPCR analysis show that COX2 expression in the heart was increased after DE exposure. The increased COX2 activity and expression likely play an essential role in mediating PGI2 production. To further confirm the specific contribution of COX1 and COX2 to the increased PGI2 production, we administered a specific inhibitor of COX1 (SC560) or COX2 (NS398), respectively. Although we found a significant change in PE-stimulated vasoconstriction in the presence of non-specific COX inhibitor (indomethacin), we failed to observe any difference of vasoconstriction in the presence of the specific antagonists of COX1 (SC560) or COX2 (NS398) (Appendix E). We speculate that there was an interaction between COX1 113  and COX2 and the inhibition of both COX1 and COX2 activities was necessary to reveal the inhibitory effect. Other possible factors, that may be responsible for the negative results and we can not exclude, include the concentration of these antagonists and the time course of administration. COX2 has been implicated in the progression of atheroslcerosis, and PGI2 can also act as a pro-inflammatory prostanoid that enhances vascular permeability and promotes leukocyte infiltration. We postulate that COX2 up-regulation contributes to the DE-exposure induced cardiovascular dysfunction. COX2 could be a source for ROS (324); induce endothelial dysfunction by attenuating NO production (325); activate leukocyte chemotaxis; and promote smooth muscle cell migration (3), all factors that promote atherogenesis. It is worth noting that COX2 is an intermediate enzyme, which is involved in the production of an array of PGs, thus COX2-mediated functions in cardiovascular system largely depend on the downstream enzymes present in a particular cell type. The evidence from clinic trials has shown that coxibs, a subclass of non-steroidal anti-inflammatory drugs designed to selectively inhibit COX2, are associated with increased cardiovascular events, including MI, and thrombotic stroke (327; 328). These side effects could result from reduced COX2-mediated PGI2 production. PGI2 is an anti-atherogenic molecular, and can protect the vasculature from inflammation and inhibit atherogenesis by reducing ROS production and inhibiting platelet activation (329). In addition, PGI2 can also cause the up-regulation of eNOS expression and activity (330). To examine whether the increased PGI2 has a beneficial effect or is just a compensatory effect, genetic modified animal models and/or PGI2 analogs could help to understand the role of COX2 and PGI2 in vascular diseases associated with exposure to DE.  114  In summary, we show that DE exposure caused up-regulation of COX2 (but not COX1) activity and expression in the blood vessels and the heart, which could contribute to DE exposure-induced cardiovascular disease.  115  Chapter 6 Exposure to Diesel Exhaust Enhances Soluble Guanylate Cyclase Expression in ApoE Knockout Mice  6.1 Introduction The association between exposure to ambient particulate matter air pollution with diameter less than 10µm (or PM10) and cardiovascular morbidity and mortality has been well established (22). However, the mechanisms underlying how PM10 impacts blood vessels are not fully understood. Abundant evidence indicates that endothelial dysfunction is an important component in the pathogenesis of PM10-associated cardiovascular diseases (22; 287). The endothelium, a monolayer of cells separating blood from the vascular wall, serves as an important regulator to maintain vascular tone and vasculature integrity (276-278). It does so by producing a number of mediators, in particular, nitric oxide (NO) and endothelin (ET). NO produced by endothelial cells can diffuse into the vascular smooth muscle cells and activates guanylyl cyclases (GC), which consequently catalyze the dephosphorylation of guanosine 5’-triphosphate (GTP) to 3’,5’-cyclic guanosine monophosphate (cGMP), and relax blood vessels. There are 2 types of GC: soluble and particulate. Soluble GC (sGC) is expressed in cytosoplasm and particulate GC is expressed in cell membrane. In general, particulate GC does not respond to NO, and soluble GC is known as the NO receptor in smooth muscle cells (331). sGC is a heterodimer composed of an α subunit and a β subunit, each of which is further characterized into two isoforms: α1, and α2, β1, and β2. The 116  expression of subunits varies among different tissue types: α1 and β1 are expressed ubiquitously in blood vessels, but α2 and β2 are not expressed in vasculature. Studies have shown that α1 subunit is essential for the basal NO and SNP-stimulated sGC activation, whereas β1 subunit is critical for sGC specificity for GTP (331). Endothelium-derived NO protects the blood vessels from developing atherosclerosis by suppressing vasoconstriction, attenuating monocyte chemotaxis, preventing the adherence of leukocytes to the endothelium, inhibiting platelet adherence and aggregation, and controlling the proliferation of vascular smooth muscle cell (279). A hallmark of endothelial dysfunction is an impairment of acetylcholine (ACh)-stimulated vasorelaxation. ACh can stimulate endothelial cell to produce NO. The loss of endothelium-derived NO bioactivity is critical to this abnormal response (165). NO is enzymatically synthesized by nitric oxide synthase (NOS) from L-arginine in the presence of co-factor terahydrobiopterin (BH4) (332). Three isoforms of NOS have been identified, including endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). Exposure to PM10 can cause insufficient supply of eNOS substrate (L-arginine), inactivation/oxidation of cofactor BH4, attenuation of eNOS activity and expression, and degradation of NO bioactivity (22; 287), consequently resulting in the impairment of AChstimulated vasorelaxation, or endothelial dysfunciton. In addition, exposure to PM10 causes excessive ET-1 production (196), enhanced vascular sensitivity to ET-1 (284), and upregulation of ET-1 mRNA expression (200), resulting in exaggerated vasoconstriction, which contribute to hypertension, MI, and stroke.Studies from our laboratory have shown that exposure to PM10 (e.g., ECH93), caused the acceleration of atherogenesis in rabbits (133; 143; 545). Sun and colleagues showed that exposure to concentrated ambient PM2.5 caused progression of atherosclerosis in ApoE knockout mice (143). Thess findings have been supported by human studies showing that increased ambient PM is associated with the 117  development of atherosclerosis (144; 145; 300). Recently, we demonstrated that exposure to diesel exhaust (DE), a major component of urban PM2.5, promotes the progression of atherosclerosis (280). In vitro and instillation studies have shown that DE exposure causes lung inflammation, and vascular endothelial dysfunction (180; 183). In the study, we examine whether DE exposure causes endothelial dysfunction, using a model of chronic inhalation exposure DE to ApoE knockout mice, and to investigate the signaling pathways that contribute to the DE exposure-induced endothelial dysfunction, and ultimately the progression of atherosclerosis.  6.2 Methods and Materials (Please refer to Chapter 2 for detailed information)  6.2.1 Methods Male ApoE knockout mice (30-week old), fed with regular chow, were exposed for 7 weeks (5days/week, 6hrs/day) to DE controlled at the concentration of 200 µg/m3 PM2.5. Mice exposed to filtered air were the control. Animal procedures were approved by the Animal Care and Use Committee of the University of Washington. After exposure, mice were euthanized. Plasma, and urine were collected and kept at – 80°C. The thoracic aorta, abdominal aorta, and heart were carefully dissected from their connective tissues and kept in appropriate solution until assay. To examine endothelial function, thoracic aortae were cut to 2mm rings and mounted on a wire myograph. The relaxant responses to acetylcholine (ACh), and sodium nitroprusside (SNP) were determined. To examine the effect of DE exposure on endogenous NO production, NG-nitro-L-arginine-methyl ester (L-NAME; 200µM) was used. In addition, a specific inhibitor for iNOS (1400W) was administered to examine the contribution of iNOS activity. The impact of DE exposure on ET-1 effect was examined using the ET  118  receptor antagonist (bosentan, 10 µM), selective inhibitor for ETA (BQ123, 10µM) or ETB (BQ788, 10µM), respectively. Cryosections (5µm) were incubated with monoclonal anti- eNOS antibody (1:50, BD Transduction Laboratories) at room temperature for 1h. Paraffin-embedded sections (5µm) were incubated with rabbit polyclonal antibodies to sGCα1 (1:200), and sGCβ1 (1:200) at 4°C overnight. The positive red staining was recognized, quantified by colour segmentation (Image Pro Plus), and normalized to the area of the atherosclerotic lesion. Plasma NO level was assessed by measuring nitrite concentration using a NO-specific chemiluminescence analyzer (Sievers). Urine cGMP level was measured using a cGMP EIA kit (Cayman Chemical) and normalized to the urine creatinine (Cr.) concentration. Total RNA was extracted from the abdominal aortae using RNeasy Fibrous Tissue Mini Kit (Qiagen). The mRNA expression of eNOS, sGCα1, β-actin, and hypoxanthine phosphoribosyltransferase-1 (HPRT1) was measured by qRT-PCR (TaqMan) using the ABI Prism 7900HT sequence detection system (Applied Biosystems). The values of gene expression were normalized to the values of β-actin, and HPRT1, and displayed as ratio relative to β-actin.  6.2.2 Solutions and Chemicals The PSS consisted of the following (in mM): NaCl 119, KCl 4.7, KH2PO4 1.18, NaHCO3 24, MgSO47H2O 1.17, CaCl2 1.6, glucose 5.5 and EDTA 0.026. All reagents were purchased from Sigma (St. Louis, MO).  6.2.3 Statistical Analysis Results are reported as mean±SEM. The statistical significance was evaluated using the unpaired Student’s t test for simple comparison between two values. The concentration119  response curves of the different groups were compared by ANOVA for repeated measurements followed by Bonferroni’s correction. P<0.05 was considered to be significant. In all experiments, n equals the number of mice from which samples were obtained.  6.3 Results  6.3.1 Endothelial Function ACh-stimulated relaxation was not impaired by DE exposure (Fig.6.1A); however, the concentration-response curve of SNP-induced endothelium-independent relaxation was significantly shifted to the left after DE exposure (pD2 (-Log [EC50]): 7.9±0.1 vs 8.5±0.1; Filtered air vs. DE, p<0.001) (Fig.6.1B), suggesting an increase in smooth muscle cell sensitivity to NO. To examine whether the increased NO sensitivity was due to the reduction of endogenous NO production, we inhibited the NO production with a NOS antagonist (LNAME). Indeed, the concentration-response curve of SNP shifted to the left in the presence of L-NAME (Fig.6.1C). To examine the effect of DE exposure on the endogenous NO production, we used PE as an agonist. In the presence of L-NAME, the fractional change of PE-induced maximal constriction was not significantly different (29.1%±3.5 vs 38.7±2.9%; Filtered air vs. DE, p=0.1) (Fig.6.2C). However, exposure to DE significantly increased the fractional change of vasconstriction in the presence of specific inhibitor for iNOS (1400W) (7.2±2.1% vs 24.1±7.6%; Filtered air vs. DE, p<0.04) (Fig.6.2F), suggesting an increase in iNOS activity, which indirectly implys a reduction in eNOS activity.  120  A  100  % Relaxation  75  50  25 Filtered Air DE  0 -9  -8  -7  -6  -5  Log[ACh](M)  B  C  **  100  100  *  75  *  *  50  * 25  Filtered Air  % Relaxation  % Relaxation  * 75 50 25  SNP SNP+L-NAME  DE  0 -10  -9  -8  Log[SNP](M)  -7  -6  0 -10  -9  -8  -7  -6  Log[SNP] (M)  Figure 6.1 Concentration-response curves show that ACh-stimulated endothelium-dependent and SNP-elicited endothelium-independent relaxation. A) Concentration-response curves show that ACh-stimulated relaxation is not different between DE and filtered air exposure groups, n=6; B) The concentrationresponse curve of SNP-induced endothelium-independent relaxation shifted to the left after DE exposure, n=6, *P<0.05; C) Inhibition of NOS by L-NAME led to a left-shiftiness of the concentration-response curve of SNP relaxation, n=6, *P<0.05. Values are mean±SEM.  121  A  20  ** *  Tension (mN)  Tension (mN)  16  * *  16  *  12  B  20  8  * 4  *  * *  *  12  * 8  * 4  PE (Filtered Air)  PE (DE)  PE+L-NAME  PE+L-NAME  0 -9  -8  -7  -6  0  -5  -9  Log[PE](M)  C Fractional Changes of T ension (%)  50  -7  -6  40 30 20 10 0 50µ µM PE+ L-NAME  E  12  8  4  8  4  PE (DE)  PE (Filtered Air)  PE+1400W  PE+1400W  0  0 -9  -8  -7  -6  -5  Filtered Air DE  Tension (mN)  Tension (mN)  -8  Log[PE]  D  12  **  -9  -5  -8  -7  -6  -5  Log[PE](M)  Log[PE](M)  Fractional Changes of Tension (%)  F 40  Filtered Air DE  *  30 20 10 0 50 µ M PE+ 1400W  Figure 6.2 The effect of NOS inhibitors on PE-induced vasoconstriciton in the thoracic aorta. Concentration-response curves show that the inhibitor of NOS (LNAME) significantly enhanced vasoconstriction in both control (A) and DE exposure groups (B) n=8, *P<0.01. However, the fractional change of maximum vasoconstriction in the presence of L-NAME was not significantly different between the control and DE exposure groups (C), n=8, p=0.10. Concentration-response curves show that the inhibitor of iNOS (1400W) did not significantly enhance vasoconstriction in both control (D) and DE group (E), n=9, p=0.11. However, 1400W caused a significant increase in fractional change of maximum vasoconstriction in DE exposure group, n=9, *P<0.04. Values are mean±SEM. 122  6.3.2 Immunohistochemical Staining of eNOS and sGC Expression in the Thoracic Aorta eNOS expression in the thoracic aorta was markedly reduced after DE exposure (76.5±8.5% vs 45.2 ± 7.5%, Filtered air vs. DE; p<0.02) (Fig.6.3, A,B,C). sGCα1 expression was significantly increased following DE exposure (1.2±0.7% vs 8.9 ± 3.7%, Filtered air vs. DE; p<0.02) (Fig.6.3, D,E,F). The expression of sGCβ1 was similar, compared DE with filtered air group (Appendix F).  Filtered Air  DE  A  B  L  L  20µm  C Filltered Air DE  Volume Fraction of e NOS Expre ssio n (V/v%) (Normalize d to the Le ng th of Endo thelium)  100 80  *  60 40 20 0 Filltered Air  DE  123  Filtered Air  DE  D  E  L  L  F Volume Fraction o f α 1 Expression (V/v%) GCα (Normalized to Vascular Wall Thickness)  15 Filtered Air DE  *  10  5  0 Filtered Air  DE  Figure 6.3 Immunohistochemical analysis of eNOS and sGCα1 expression in the thoracic aorta. A,B) Representative photomicrographs of staining for eNOS in the thoracic aorta; C) Quantitative analysis shows that DE exposure significantly reduced eNOS expression in the thoracic aorta , n=9, *P<0.02; D,E) Representative photomicrographs of staining for sGCα1 in the thoracic aorta; the inset in E) is a photomicrograph with higher magnification (600X); F) Quantitative analysis shows that exposure to DE enhanced sGCα1 expression in the thoracic aorta, n=9, *P<0.02. L: Lumen. Magnification: orginalX400. Values are mean±SEM.  124  6.3.3 Measurement of Plasma Nitrite and Urine cGMP Concentration Plasma nitrite concentration was not different between filtered air and DE exposure groups (Fig.6.4A). Urine cGMP concentration was higher in DE group, but the difference was not statistically significant (Fig.6.4B).  A  Filtered Air DE  800  cGMP/Cr (pmol/mg)  60  Nitrite(µ µ M)  B  40  20  600  Filtered Air DE  400 200 0  0 Filtered Air  DE  Filtered Air  DE  Figure 6.4 Measurement of plasma nitrite concentration and urine cGMP level. A) Plasma nitrite (NO metabolite) concentration was similar between filtered air and DE exposure group, n=12; p=0.7; B) Urine cGMP level was elevated after DE exposure (n=10), but the increase is not statistically significant from the control (n=9), p=0.4. Values are mean±SEM.  125  6.3.4 Real-Time RT- PCR of eNOS and sGCα1 mRNA Expression RNA was extracted from the abdominal aorta to examine whether the mRNA expression of eNOS and sGCα1 was altered after DE exposure by real-time PCR using Taqman assay. Exposure to DE reduced eNOS mRNA expression (73.8±4.7% vs 67.8± 2.7%, Filtered air vs. DE; p=0.2) (Fig.6.5A), but the reduction did not reach statistical significance. Similar, the expression of sGCα1 was not significantly different, compared DE with filtered air group (Fig.6.5B).  B  100  Filtered Air DE  Relative GC mRNA Expression to β -actin (%)  Relative eNOS mRNA Expression to β -actin (%)  A  80 60 40 20 0  Filtered Air  DE  100  Filtered Air DE  80 60 40 20 0  Filtered Air  DE  α1 in the abdominal aorta. A) Figure 6.5 The mRNA expression of eNOS and sGCα The mRNA expression of was not significantly different, compared DE exposure (n=7) with the filtered air (n=10), p=0.2; B) The mRNA expression of sGCα1 was not significantly different after DE exposure (n=8), compared with filtered air exposure (n=10), p=0.2. Values are mean±SEM.  126  6.4 Discussion In vitro and instillation studies have reported that exposure to PM10 (e.g., ECH93, residual oil fly ash, and DE), induced endothelial dysfunction (104; 177; 280); however, we did not observe a blunted ACh relaxation following a 7-week DE exposure to ApoE knockout mice (Fig.6.1A). Our finding is consistent with the results reported by Sun et al showing that exposing concentrated ambient particles to ApoE mice fed with regular chow did not impair ACh relaxation (143). In addition, a recently published inhalation study also showed that exposure of ApoE mice fed with regular chow to DE for 5 months did not attenuate ACh relaxation (297). The discrepancies could be due to the composition of different particles (ECH93 vs. DE), the size of the particles (PM10 vs. PM2.5 vs. PM0.1), and the route of exposure (in vitro vs. instillation vs. inhalation). In addition, the impairment of ACh relaxation was mostly observed after acute or subchronic exposure to PM10, the diminished impairment of ACh relaxation following chronic exposure may result from a compensatory effect. As no specific eNOS antagonists are commercially available, we used inhibitors for NOS and iNOS to examine eNOS activity indirectly. We found no significant difference of NOS activity using the non-specific NOS antagonist, L-NAME (Fig.6.2A); however, there was a significantly increased fractional change in PE-stimulated constriction in the presence of iNOS inhibitor (Fig.2B), suggesting an increase in iNOS activity, and indirectly indicating a reduction in eNOS activity, based on the observation that iNOS and eNOS are the most predominant NOS isoforms in mouse aorta (559). The result, implying suppressed eNOS activity, is supported by the immunohistochemistry analysis showing that DE exposure caused significant attenuation of eNOS expression in the thoracic aorta (Fig.6.3C). Taken together, the data, showing no impairment of ACh relaxation, but reduced endogenous eNOS-derived NO production and eNOS expression in the blood vessels, imply that a 127  “helper” mechanism(s) was turned on to compensate the reduction in endothelium-generated NO, whereby maintaining ACh-stimulated vascular relaxation after exposure to DE. Clonidine can increase endothelial NO production and induce endothelial-dependent NO-mediated vasodilatation via the activation of endothelial α2-adrenoceptors (560). To determine a potential contribution of endothelial α2-adrenoceptors to the normality of ACh relaxation after exposure to DE, we examined vasorelaxation to clonidine. The clonidineinduced vasorelaxation was similar between DE and filtered air group (Appendix G), indicating that DE exposure did not have effect on the α2-adrenoceptor-mediated response. It was reported that plasma ET-1 concentration was elevated after exposure to ambient PM (561; 196). In addition, ET-1 expression in the blood vessels, and vascular sensitivity to ET-1 were also enhanced following DE exposure (284; 336). ET-1 causes vasoconstriction after binding to the ETA receptor in smooth muscle cells. The increased ET-1 production is believed to contribute to hypertension, MI and stroke associated with exposure to ambient PM. Nevertheless, ET-1 can also cause endothelial-dependent NO-regulated relaxation by binding to the ETB receptor in endothelial cells (113). Thus, we administered both nonselective ET receptor antagonist (bosentan), and selective antagonists for ETA (BQ123) or ETB receptors (BQ788), receptively, to examine the effects of DE exposure on different ET receptors. But we did not observe any difference of vascular constriction in the presence of different ET receptor antagonists (Appendix H), suggesting that exposure to DE had no effect on ET. Interestingly, DE exposure increased SNP-induced NO-mediated endotheliumindependent vasorelaxation. It was reported that blocking basal NO production by NOS inhibitor or removing the endothelium from a segment of blood vessel resulted in augmented SNP relaxation (338). This notion was supported by in vivo study showing that SNP relaxation was enhanced in eNOS knockout mice (339). Consistent with these results, we 128  also found that after inhibition of NOS activity with L-NAME, SNP relaxation was enhanced in the thoracic aorta of ApoE knockout mice (Fig.6.1C), suggesting that the reduced basal NO production contributed to the augmented SNP relaxation. The mechanisms of the increased sensitivity to nitrovasodilators after removal of endothelium-derived NO have been investigated (340-342). Moncada and colleagues showed that the increased sensitivy was regulated at the sGC level, because the vasodilator actions of 8-bromo-cGMP was not affected (338). In this study, we showed that exposure to DE increased sGCα1 expression in the blood vessels (Fig.6.3F). PM10 can up-regulate sGC activity by direct interaction with the blood vessels (343), or/and by direct interaction with DE constituents (33). We recently reported that exposure to DE induced peroxynitrite production, which can also enhance sGC activity (344). Nevertheless, how ambient DE interacts with sGC is not clear. To examine whether the potentiated SNP response was derived from increased cGMP production, we measured levels of cGMP, and observed no elevation of urine cGMP in DE exposed mice, compared with the controls. This result suggests that the enhancement of SNP relaxation was predominately mediated at the NO receptor level, or via the up-regulation of sGC expression. We speculate that the suppressed basal endothelium-derived NO production, which occurred as a consequence of decreased eNOS expression, increased sGC activity and expression. The negative feedback-mediated sGC up-regulation could act as a compensatory effect maintaining a normal vascular function after exposure to DE. However, further investigation is needed to determine how this alteration of NO level regulates sGC activity in the blood vessels. Moreover, the mRNA expression of eNOS and sGCα1 was not affected by DE exposure (Fig.6.5), implying that DE exposure-induced reduction in eNOS expression and the up-regulation of sGC expression were regulated at post-translational levels. 129  In summary, we demonstrate that eNOS activity and expression were suppressed after DE exposure. However, ACh-stimulated endothelium-dependent relaxation was not impaired. We postulate that the up-regulation of sGC activity and expression in smooth muscle cells contribute to the unchanged ACh relaxation.  130  Chapter 7 Summary and Conclusions  Exposure to PM10 is an independent risk factor for cardiovascular events (22). The WHO estimates that air pollution is responsible for approximately three million premature deaths  each  year  (World  Health  Organization  (2002)  World  Health  Report.  [http:www.who.int/whr/2002/en]). Because of its ubiquitous and involuntary nature, exposure to PM10 may continuously enhance cardiovascular risk among millions of susceptible people worldwide in an often inconspicuous manner. Beyond serving as a simple trigger, PM10 also elicits numerous adverse biological responses (such as systemic inflammation) that may augment future cardiovascular risk after months or years of exposure. Among numerous constituents of PM10, diesel exhaust particles (DEP) are the single biggest contributor, accounting for up to 90% of the PM10 in many cities around the world. Still, the effect of exposure to DE on cardiovascular dysfunction is not well understood. In this dissertation, the impact of exposure to DE on atherogenesis and the mechanisms underlying DE-induced progression of atherosclerosis were studied. Using a well-controlled inhalation model, this study has overcome some significant shortcomings of the population-based and instillation studies, especially in the inaccurate measurement of PM10 levels, and in the less established exposure approach that may not truly represent the exposure in the real-world exposure. This dissertation provides new insights into the mechanisms underlying the progression of atherosclerosis induced by exposure to DE. By understanfing the biological signaling pathways, and using appropriate medical prevention, more immediate and effective 131  strategies may be developed to alleviate DE-inudced cardiovascular mortality and morbidity. In addition, this work provides further evidence that can be used to influence policy, to ultimately reduce ambient particles levels, instead of focusing mainly on treating the symptoms and the cardiovascular diseases induced by exposure to PM.  7.1 Relationship between DE Inhalation and Atherogenesis  In this study, ApoE knockout mice, a well-established mouse model for atherosclerosis, were exposed to DE at environmentally relevant levels for 7 weeks (6h/day, 5day/week). Using a quantitative morphometric analysis, the data in Chapter 3 show that DE exposure results in compositional changes in atherosclerotic plaques (such as increased lipid content, foam cell formation, and smooth muscle cell content), which are associated with plaque vulnerability. This study provides new information that, in addition to PM10 and concentrated ambient particles, the exposure to DE for a relatively short period of time also promotes atherogenesis. Our results also show that DE is more active or potent in promoting ambient particle-associated atherogenesis, in comparision to the results from another inhalation study showing that ApoE mice needed a 6-month exposure to concentrated ambient particles to cause the progression of atherosclerosis. Systemic inflammation is well established to play a key role in PM10-associated cardiovascular dysfunction. We counted blood smear band cells, a marker of bone marrow stimulation, and found they were significantly increased after DE exposure (Fig.3.2A). In addition, the increased band cells were positively correlated with alveolar macrophages in the lung (Fig.3.2B), indicating an association between the magnitude of DE exposure and the systemic response. In addition, a strong association was seen between the magnitude of particle deposition in the lung and the compositional changes in atherosclerotic plaques, (Fig.3.6A), suggesting a connection between exposure to DE and the vascular consequences. 132  These findings are in accordance with the results found in Watanabe heritable hyperlipidemic rabbits, where accelerated atherosclerotic lesions were positively correlated with the number of alveolar macrophages that phagocytosed PM10 in the lung and the increase in circulating band cells (141). DE exposure increased the expression of a range of surrogate markers for ROS in aortic root, including iNOS, CD36, and NT (Fig.3.5). Moreover, systemic lipid and DNA oxidation were also increased after exposure to DE (Fig.3.7). The correlation between these ROS markers and compositional changes of atherosclerotic plaques (Fig.3.6 B,C) supports the idea that ROS is involved in the progression of atherosclerosis induced by DE exposure. Collectively, these results demonstrate that exposure of ApoE knockout mice fed with regular chow to DE change the composition of atherosclerotic plaques toward a more unstable phenotype. We also show that increased oxidative stress was involved in the DE-induced deleterious effect. We did not examine the sources for DE-induced ROS produciton; however, a number of in vitro studies have shown that the NADPH oxidases, mitochondrial sources, and cytochrome P450 enzymes contribute to ambient PM-activated ROS-generating pathways (346-348). To untangle the ROS-generating pathways that are activated by exposure to DE, future ex vivo and/or in vitro experiments, incubating tissue cells (such as alveolar macrophages, epithelial cells, or isolated blood vessels) with DE collected from the filters may be worth investigating.  7.2 Mechanisms Underlying DE Exposure-induced Atherogenesis  7.2.1 Increased iNOS Expression iNOS is capable of generating superoxide (299), which can cause modification of LDL leading to the acceleration of lipoprotein-uptake by macrophages and the formation of foam cells (310), a critical step in the development of atherosclerosis. The addition of oxygen free 133  radicals generated by iNOS can impair endothelial function by attenuation of eNOS activity and the expression and/ or through direct inactivation of NO (249), resulting in endothelial dysfunction and development of atherosclerosis. In addition, iNOS and/or its-mediated reactive oxygen species have been reported to modulate matrix degradation, to contribute to the degradation and reorganization of the extracellular matrix (ECM), thus increasing the instability of atherosclerotic plaque (350; 351). Studies have shown that exposure to PM upregulates iNOS expression in the lung. Here we measured iNOS activity and expression at both protein and mRNA levels using vascular function and molecular biology approaches. The study presented in Chapter 4 demonstrates that DE exposure attenuated vasoconstriction, which was partly due to the enhancement of iNOS activity (Fig. 4.1). In addition, we showed that exposure to DE upregulated iNOS at both protein and mRNA levels in aorta and heart tissue (Fig.4.2 & Fig.4.5A). The positive correlation between increased iNOS expression in atherosclerotic plaque and increased band cells (Appendix I), indicates a systemic inflammatory responseregulated reaction. The correlation between increased iNOS expression and alveolar macrophages, and the association between increased iNOS expression and foam cell formation in atherosclerotic plaques (Fig.7.4B) also suggests that DE-induced up-regulation of iNOS plays an important role in the progression of atherosclerosis. The expression of iNOS that co-localized with CD36 and NT (Fig.4.4) implies a ROS-mediated up-regulation of iNOS expression and activity, mediated by NF-κB activation (Fig.4.6). Collectively, we show that exposure to DE up-regulates iNOS activity and expression, which could be critically important in the progression of atherosclerosis induced by DE exposure. To validate the notion that up-regulation of iNOS by DE exposure contributes to atherogenesis, we may investigate the exposure of iNOS/ApoE double knockout mice to DE. In addition, exposure iNOS/ApoE double knockout mice to DE with administering the 134  specific iNOS antagonist (1400W) may provide supportive information to understand the pivotal roles of iNOS in DE exposure-induced atherogenesis.  7.2.2 Increased COX2 Expression Inflammation is characteristic of all the phases of atherosclerosis progression, from the initial formation of the early lesions to the rupture of advanced plaques, at both systemic and local levels. Cyclooxygenase (COX), a key enzyme in the conversion of arachidonic acid to prostanoids, has some potential molecular mechanisms that may contribute to atherogenesis and atherosclerotic plaque instability (273; 352). Additional evidence led us to examine the COX pathway with the iNOS blocker, 1400W, which did not completely restore the suppressed PE-elicited vasoconstriction, though, in the presence of both COX and iNOS blockers, the attenuated PE-stimulated vasoconstriction after exposure to DE was completely restored (Appendix J). This indicates that the exposure to DE enhanced both COX and iNOS activities. The study presented in Chapter 5 shows that exposure to DE increased COX activity (Fig. 5.2), predominantly due to the up-regulated expression of COX2, but not of COX1, in the vascular wall (Fig.5.4). In addition, we showed that the expression of COX2, but not COX1, was enhanced in atherosclerotic lesions in aortic root (Fig.5.6). Moreover, the mRNA expression of COX2 was increased (Fig.5.8A,B). We also correlated COX2 expression with both the magnitude of DE exposure (indicated by total alveolar macrophages in the lung and the alveolar macrophages positive for particles) and systemic response (indicated by band cells) to determine the relationship between COX2, the lung effect and the systemic response, but no significant association was found. This suggests that COX expression in atherosclerosis plaque following DE exposure was mediated locally in vascular walls. iNOS has been reported to cause up-regulation of COX, especially under pathological 135  conditions (353; 354). We observed that the expression of COX2 and iNOS by macrophages and smooth muscle cells are co-localized in blood vessels (Appendix J). We speculate that iNOS contributes to the up-regulation of COX2 locally. The pathway(s) leading to COX activation by NO/iNOS are unclear, but may involve an interaction at the iron-heme center of COX molecule where NO interacts with iron- containing enzymes leading to either a stimulation (such as via soluble guanylate cyclase) or inhibition (such as via aconitase) of the enzymatic activity (355; 356). This interaction between NO and COX activation may be a critical determinant in the optimal functioning of COX. For example, in pathological conditions such as inflammation, where both COX and iNOS pathways are augmented, regulation of COX activity by NO/iNOS may represent an important mechanism by which the initial inflammatory response can be amplified. Collectively, we show that exposure to DE enhances COX2 (but not COX1) expression, possibly by interacting with inflammatory mediators, such as iNOS. Nevertheless, the interaction between COX2 and iNOS activation and the way in which how COX2 up-regulation is related to DE-induced vascular abnormality, warrants further investigation.  7.2.3 Vascular Endothelial Function The endothelium consists of a monolayer of endothelial cells, and is located at the interface between the blood and the vessel wall. In addition to physiologically protecting the interaction between blood cell and the vessel wall, the endothelium also secretes a number of vasoactive substances (such as NO and ET-1) to regulate vascular smooth muscle tone, and prevents atherogenesis by reducing coagulation, leukocyte adhesion, and vascular smooth muscle cell proliferation and migration. A number of in vitro experiments have shown that exposure to PM10 induces endothelial dysfunction (104; 143; 177; 280; 358). 136  The study presented in Chapter 6 delineates the effect of DE exposure on endothelial function. We found that exposure to DE did not blunt ACh relaxation of the thoracic aorta (Fig.6.1A). Nevertheless, indirect evidence from the vascular function study, using NOS and iNOS blockers, respectively, implied that endogenous NO production was suppressed after exposure to DE (Fig.6.2). An immunohistochemical analysis showed that the expression of eNOS in the thoracic aorta was attenuated (Fig.6.3C). Interestingly, the concentrationresponse curve of SNP-induced endothelial-independent relaxation was shifted to the left (Fig.6.1B), suggesting an increase in smooth muscle sensitivity to NO, which could be explained by enhanced sGC expression or/and increased cGMP production in vascular smooth musclce cells. Indeed, we observed that DE exposure enhanced the sGCα1 expression (Fig.6.3F), though the elevation of cGMP production was not statistically significant (Fig.6.4B). We have also found that iNOS was up-regulated after DE exposure, but found that ACh relaxation was not affected in the presence of the iNOS blocker (1400W) (Appendix K), indicating that iNOS did not contribute to this unchanged ACh relaxation response. De and colleagues showed an interaction between NO and PGI2, and that the release of these two endothelial relaxants are coupled (359). COX2 is recognized as the predominant mediator of PGI2 production. We speculate that the COX2-mediated increase of PGI2 may contribute to the intact ACh relation as a compensatory effect. Our finding of an increased PGI2 expression in blood vessels and an elevation of PGI2 metabolite in mouse urine, suggests the involvement of PGI2 in ACh-relaxation after DE inhalation. Collectively, exposure to DE attenuated the eNOS expression in blood vessels, but the ACh-induced relaxation was not affected. This phenomenon could be due to compensatory effects from the up-regulated expression of sGC and PGI2. The interactions among these endothelium mediators need to be further delineated.  137  In summary, our study significantly extends previous data showing that PM10 potentiates atherosclerotic lesion development in animals (141; 143). I have demonstrated that exposure to DE at an environmentally relevant concentration causes progression of atherosclerosis in ApoE knockout mice fed with regular chow. Our results support the notion that the generation of systemic oxidative stress and up-regulation of iNOS are responsible for the observed vascular effects. Our data also indicate an interaction between iNOS and COX2 induced by exposure to DE. In addition, compensatory reactions may also be engaged after exposure to DE, in maintaining vascular homeostasis.  138  Exposure to DE  Lung inflammation  Systemic response  Bone marrow stimulation with the release of leukocytes and platelets  Oxidative stress iNOS COX2 eNOS  Endothelial cell injury NF-κB activation  Foam cell formation Smooth muscle migration Lipid content  Progression of atherosclerosis Instable plaque  Other insults Acute vascular events MI and Stroke Figure 7.1 Summary of the signalling pathways contributing to DE inhalationinduced atherogenesis  139  7.3 Limitations: 1. DE has an odor, and it might not be well controlled in the filtered air (control) exposure because the intermission of exposure to DE and filtered air exposures was less than 2 weeks, and the odor in exposure facility might not be cleared by HEPA, and potential effects of the odor was not addressed. 2. Although ApoE knockout mice is an established model for atheroclerosis, plaque rapture, which is considered to be pathogenesis of MI, is rarely observed in this mouse model, and this fact may restrict the possibilities of examining and approving the hypothesis: DE causes plaque rapture, hence lead to MI, and stroke. 3. To confirm the causal effect of exposure to DE and up-regulation of iNOS and COX2, transgenic mice would be needed. 4. The exposure protocol used in this study was well controlled and freshly generated. The lab exposure may not fully represent real-life diesel exhaust exposure. For example, people may be exposed to diesel exhaust that was not freshly (less than 10min) generated, but produced by these cars miles away and travelled in the air for a while. The aged diesel exhaust may have different composition comparing with freshly generated diesel exhaust, hence the impact of aged and freshly generated diesel exhaust on the blood vessels could be different.  7.4 Future Directions  In this dissertation, in addition to demonstrating DE inhalation-induced compositional modification of atherosclerotic plaque, I examined three signaling pathways that may be responsible for the progression of atherosclerosis in response to DE inhalation. Our results have also led to a number of questions; in particular how the three closely related signaling cascades (iNOS, COX, and eNOS) interact with each other. Transgenic animal models 140  (deficient in iNOS, COX, or eNOS genes) may be useful for clarifying the interactions of these enzymes. Clearly, a better understanding of the mechanisms would be valuable for future interventions to decrease the impact of exposure to particulate matter air pollution, considering the dramatic effect on cardiovascular morbidity and mortality. Because endothelial dysfunction can occur before the symptoms of cardiovascular diseases appear, it is particularly imperative to know how the processes involved in NO synthesis and availability are affected and how these changes can be prevented or therapeutically treated. Also, it is important to learn how changes in NOS and COX activities are related to other outcomes of PM exposure, such as changes in heart rate variability, autonomic disturbances, and arrhythmias. Our findings also support the observations from a number of epidemiological studies, that pre-existing pathological conditions accelerate the progression of atherosclerosis. More work is required to establish how susceptibility-enhancing states (high cholesterol, smoking, and hypertension) can promote PM10 toxicity or cardiovascular exacerbations. The result of such studies may also suggest mechanisms that determine cardiovascular disease vulnerability and risk that could be used in designing urgently needed epidemiological studies to determine which specific sub-populations may be more sensitive to the effects of PM10. Moreover, studies are required to interpret some important questions derived from epidemiological observations, such as lag effects, dose-response effects, and the possibility of a threshold level for igniting a cardiovascular effect. Furthermore, an acute exposure model may be necessary to reveal the mechanisms underlie DE-induced acute cardiovascular events.  141  Bibliography 1. Nemery B, Hoet PH, Nemmar A. The Meuse Valley fog of 1930: an air pollution disaster. Lancet 2001;357:704-708 2. LOGAN WP. 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Proc.Natl.Acad.Sci.U.S.A 1988;85:2334-2338  168  APPENDIX A  % Proteoglycan/Plaque Area  Expression of Proteoglycan in Aortic Root Lesion  50 40 30 20 10 0 Filtered Air  DE  The expression of proteoglycan in aortic root lesion was not affected by exposure to DE.  169  APPENDIX B  Thickness of Smooth Muscle layer  Effect on the Thickness of Smooth Muscle in Thoracic Aorta  150  100  50  0  Filtered Air  DE  Exposure to DE had no effect on the thickness of smooth muscle of thoracic aorta.  170  APPENDIX C  Association between iNOS Expression and the Cellular Features of Atherosclerotic Plaque in Aortic Root  Root Foam Ce lls (V/v %)  40  y = 0.3396x + 4.2063 R2 = 0.6836  30 20 10 0 0  20  40  60  80  Root Actin Expr ession (V/v %)  Root iNOS Expression (V/v%)  60  y = 0.2826x + 8.549 R2 = 0.3333  40  20  0 0  20  40  60  80  Root iNOS Expre ssion (V/v%)  There is a positive correlation between iNOS expression with both foam cell formation (p<0.001) and the number of smooth muscle cells (p<0.02) in atherosclerotic plaques in aortic root.  171  APPENDIX D No Changes of Vasoconstriction in the Presence of TXA2 Inhibitor  Tension (mN)  9  6  3  PE PE+SQ29548 0 -9  -8  -7  -6  -5  Log[PE] (M)  PE-concentration response curve shows that vasoconstriction of the thoracic aorta in DE exposure group was not different in the presence of TXA2 inhibitor (SQ29548).  172  APPENDIX E  Vascular Function Analysis of COX1 and COX2 Activity  A  B  12  8  4  PE (Filtered Air)  Tension (mN)  Tension (mN)  9  6  3  PE (DE)  PE+ SC560 0 -9  -8  -7  -6  PE+SC560  -5  0 -9  Log[PE](M)  -8  -7  -6  -5  Log[PE](M)  C  D 9  Tension (mN)  Tension (mN)  9  6  3  6  3  PE (DE)  PE (Filtered Air) PE+ NS398 0  PE+NS398 0  -9  -8  -7  Log[PE](M)  -6  -5  -9  -8  -7  -6  -5  Log[PE] (M)  To examine the effect of DE exposure on COX1 and COX2 activity, the specific antagonists for COX1 (SC560) and COX2 (NS398) were administered, respectively. Concentrationresponse curves show that PE-elicited vasoconstriction was not different in the presence of these antagonists, comparing DE exposure and the control. n=6. Values are mean±SEM.  173  APPENDIX F  Volume Fraction of GCβ 1 Expression (V/v%)  No Modification of DE Exposure on the Expression of sGCβ β1 in the Thoracic Aorta  4 3 2 1 0  Filtered Air  DE  The expression of sGCβ1 in the thoracic aorta was not different after exposure to DE compared with the control.  174  APPENDIX G  Concentration-Response Curve of Clonidine-Induced Vasorelaxation  100  % Ralaxation  75  50  25  Filtered Air DE 0 -9  -8  -7  -6  -5  Log[Clonidine] (M)  Concentration-response curves show that clonidine-induced vasorelaxation was not different, compared DE exposure with the control. n=6. Values are mean±SEM.  175  APPENDIX H  Vascular Function Analysis of Endothelin-1 Activity  A  B 7.5  Tension (mN)  Tension (mN)  12  8  4  5.0  2.5  PE (Filtered-air)  PE (DE)  PE+ BQ123 0 -9  -8  -7  -6  -5  PE+BQ123 0.0 -9  -8  Log[PE](M)  C  -6  -5  D  9  Tension (mN)  Tension (mN)  12  -7  Log[PE](M)  8  4  6  3  PE (Filtered Air)  PE (DE)  PE+ BQ788  PE+BQ788 0  0 -9  -8  -7  Log[PE](M)  -6  -5  -9  -8  -7  -6  -5  Log[PE](M)  To examine the effect of DE exposure on endothelin-1 (ET-1) activity, the specific antagonists for ETA (BQ123) and ETB receptors (BQ788), were administered, respectively. Concentration-response curves show that PE-elicited vasoconstriction was not different in the presence of these antagonists, compared DE exposure with the control. n=6. Values are mean±SEM.  176  APPENDIX I  Root iNOS Expression (V/v%)  Correlation between iNOS Expression and Band Cells  y = 3.3194x - 11.628 R2 = 0.7883 P=0.04  80 60 40 20 0 0  5 10 15 20 Blood Smear Band Cell Counts (%)  25  There is a positive correlation between increased iNOS expression in atherosclerotic plaque of aortic root and increased circulating band cells.  177  APPENDIX J  Fractional Change of Vasoconstriction  Vascular Function Analysis and Immunohistochemistry Analysis of COX and iNOS Activity and Expression  0.4 0.3 0.2  Filtered Air DE  0.1 0.0 Filtered Air  DE  The attenuated PE-stimulated vasoconstriction after exposure to DE was completely restored in the present of both COX and iNOS blockers. n=6. Values are mean±SEM.  A  B  Representative photomicrographs of staining for iNOS (A) and COX2 (B) (400X) that were co-locally expressed by macrophages and smooth muscle cells in the thoracic aorta..  178  APPENDIX K  Vascular Function Analysis of the Effect of iNOS on ACh Relaxation  % Relaxation  100  75  50  25  ACh ACh+1400W  0 -9  -8  -7  -6  -5  Log[ACh] (M)  Concentration-response curves show that ACh-induced endothelium-dependent vasorelaxation was not altered by the iNOS blocker (1400W), compared DE with filtered air exposure. These experiments were done using the thoracic aorta of ApoE mice exposed to DE. n= 6. Values are mean±SEM.  179  

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