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The role of peri-transplant ischemia and reperfusion injury in cardiac allograft vasculopathy Hunter, Arwen Leigh 2008

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THE ROLE OF PERI-TRANSPLANT ISCHEMIA AND REPERFUSION INJURY IN CARDIAC ALLOGRAFT VASCULOPATHY  by  ARWEN LEIGH HUNTER B.Sc., The University of Victoria, 2002  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES  (Pathology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  March 2008 © Arwen Leigh Hunter, 2008  Abstract Heart transplantation is often the only therapeutic option for patients with end stage heart disease. Allograft organs are in short supply. Thus, preserving the life of a grafted organ is extremely important. Cardiac allograft vasculopathy (CAV) is an expression of chronic rejection that accounts for the greatest loss of graft function in transplanted hearts. Peri-transplant ischemia/reperfusion (I/R)-injury occurs during transplantation when blood flow is stopped to remove the heart from the donor and then is reinstated upon implantation of the donor heart into the recipient. This oxidative injury contributes to vascular dysfunction and CAV. In this dissertation, I hypothesize that prevention and/or reduction of UR during transplantation reduces post-transplant vascular dysfunction and CAV. In this regard, myself and my colleagues, examined the roles of apoptosis repressor with caspase recruitment domain (ARC) and cytochrome p450 (CYP) 2C enzymes in UR-induced vascular dysfunction and CAV. ARC expression was detected in endothelial cells (ECs) and smooth muscle cells (SMCs); however, increased levels of ARC do not protect against oxidant injury. ARC overexpression did protect against oxidant-induced cell death in H9c2 rat embryonic myoblasts. We observed that ARC-overexpression prevented H9c2 differentiation into muscle cells. With our focus on vascular injury, we turned our attention to the CYP 2C enzymes. Both endothelium-dependent and independent vascular function was impaired following I/R. Pre-treatment with the CYP 2C inhibitor sulfaphenazole (SP) restored endothelial sensitivity to acetylcholine, but did not restore sensitivity to endotheliumindependent vasodilators. Rat heterotopic heart transplants were performed with rats being ii  treated with SP or vector control prior to surgery. Rats treated with SP showed significantly reduced luminal narrowing and had decreased SMC proliferation, oxidant and interferon-y levels. No differences were detected in immune infiltration or apoptosis. Complementary studies in cultured vascular cells revealed that CYP 2C9 expression decreased viability and increased ROS production following hypoxia and re-oxygenation in ECs but not in SMCs. In summary, we did not detect protection of vascular cells by ARC, but did discover a novel role for ARC in differentiation. CYP 2C contributes to post-ischemic vascular dysfunction and CAV through increased oxidative stress and endothelial dysfunction.  iii  Table of Contents  Abstract^ Table of Contents ^ List of Tables ^ List of Figures^ List of Symbols, Abbreviations and Acronyms ^ Acknowledgements ^ Co-Authorship Statement ^ Chapter 1: Introduction ^  ii  iv vii viii xii xiv 1  1.1 Cardiac Transplantation ^ 1 1.1.1 Historical perspective ^ 1 1.1.2 Hyperacute and acute forms of cardiac transplant rejection ^ 5 1.1.3 Chronic cardiac transplant rejection by cardiac allograft vasculopathy (CAV) ^ 9 1.1.4 Vasomotor function following transplantation ^ 16 1.1.5 Animal models of CAV ^ 18 1.2 Ischemia and Reperfusion (I/R) Injury ^ 20 1.2.1 Reactive oxygen and nitrogen species in I/R ^ 21 1.2.2 I/R and transplantation ^ 22 1.3 Apoptosis Repressor with Caspase Recruitment Domain (ARC) ^ 24 1.3.1 ARC in I/R injury ^ 25 1.4 Cytochrome p450 Enzymes (CYPs ^ 26 1.4.1 CYP 2C enzymes ^ 27 1.5 Arachidonic Acid (AA) Metabolism ^ 36 1.5.1 AA metabolism by cyclooxygenase ^ 38 1.5.2 AA metabolism by lipoxygenase ^ 38 1.5.3 AA metabolism by CYPs ^ 39 1.5.4 AA metabolites and cardiovascular disease ^ 40 1.6 Thesis objectives and hypotheses ^ 41 1.7 Bibliography ^ 43  Chapter 2: Apoptosis Repressor with Caspase Recruitment Domain in Vascular Cell Death and Myogenic Differentiation 58 ) ^  2.1 Introduction ^ 58 2.2 Aim ^ 59 2.3 Materials and Methods ^ 60 2.3.1 Cell culture ^ 60 2.3.2 RNA extraction and reverse transcriptase (RT)-PCR ^ 61 2.3.3 Cell lysis and Western blotting ^ 62 2.3.4 TAT protein expression, purification and Texas red staining ^ 63 2.3.5 TAT-fusion protein transduction and detection ^ 65 2.3.6 Cell viability^ 66 2.3.7 H9c2 stable and transient transfection ^ 67 2.3.8 H9c2 myocyte differentiation ^ 68 iv  2.3.9 F-actin and nuclear staining of H9c2 cells ^ 68 2.3.10 DEVDase activity assay ^ 69 2.3.11 Statistical analysis ^ 69 2.4 Results ^ 70 2.4.1 Native ARC expression in endothelial and smooth muscle cell lines ^ 70 2.4.2 TAT-ARC purification and transduction in vascular cells ^ 70 2.4.3 ARC over-expression does not protect against H202 treatment. ^ 73 2.4.4 Functional overexpression of ARC in pre-differentiated H9c2 cells ^ 73 2.4.5 Characterization of H9c2-ARC cell differentiation ^ 77 2.4.7 Caspase-3 activation during H9c2 differentiation ^ 82 2.5 Discussion ^ 87 2.6 Bibliography ^ 92 Chapter 3: Cytochrome p450 2C Contributes to Post-Ischemic Vascular Dysfunction 2 94 3.1 Introduction ^ 94 3.2 Aim ^ 95 3.3 Materials and Methods ^ 96 3.3.1 Heart perfusion and vessel cannulation ^ 96 3.3.2 Vasomotor responses in septal arteries ^ 97 3.3.3 Dihydroethidium (DHE) staining of coronary blood vessels ^ 97 3.3.4 Measurements of dityrosine in coronary effluents ^ 98 3.3.5 Statistical analysis ^ 98 3.4 Results ^ 98 3.4.1 Endothelium-dependent vasomotor responses ^ 98 3.4.2 Endothelium-independent vasomotor responses ^ 100 3.4.3 Post-ischemic ROS production ^ 100 3.5 Discussion ^ 107 3.6 Bibliography ^ 111 Chapter 4: Cytochrome p450 2C Contributes to Cardiac Allograft Vasculopathy 3 .... 113 4.1 Introduction ^ 113 4.2 Aim ^ 115 4.3 Materials and Methods ^ 115 4.3.1 Heterotopic heart transplantation ^ 115 4.3.2 Tissue collection ^ 116 4.3.3 Histological staining and immunohistochemistry (IHC) ^ 117 4.3.4 Histological assessment and quantification ^ 118 4.3.5 Luminex analysis ^ 118 4.3.6 8-Isoprostane measurements ^ 119 4.3.7 Statistical analysis ^ 121 4.4 Results ^ 121 4.4.1 Post-surgical morbidity and mortality ^ 121 4.4.2 Assessment of CYP 2C6 expression in rat heart cross-sections ^ 122 4.4.3 CYP 2C contributes to luminal narrowing in rat heterotopic heart transplants 122 4.4.4 Assessment of general immune infiltration ^ 122 4.4.6 CYP 2C does not significantly alter post-transplant apoptosis ^ 129 4.4.7 CYP 2C contributes to SMC proliferation following transplantation ^ 137 4.4.8 Peripheral cytokine and chemokine levels following transplantation ^ 137  4.4.9 CYP 2C contributes to serum 8-isoprostane levels ^ 142 4.5 Discussion ^ 142 4.6 Bibliography ^ 145 Chapter 5: Cytochrome p450 2C9 in Vascular Cell Death and Oxidative Stress 4 ^ 148 5.1 Introduction ^ 148 5.2 Aim ^ 149 5.3 Materials and Methods ^ 150 5.3.1 Cell culture ^ 150 5.3.2 Cell lysis and Western blotting for CYP 2C9 ^ 150 5.3.3 Adenoviral expression of CYP 2C9 in HUVEC ^ 151 5.3.4 Optimization of hypoxic conditions ^ 151 5.3.5 Cell viability in response to H/R ^ 152 5.3.6 Measurements of 8-isoprostane ^ 152 5.4 Results ^ 153 5.4.1 Native, adenoviral, and H/R-induced expression of CYP 2C9 in HUVECs.... 153 5.4.2 CYP 2C9 expression contributes to post H/R cell death in HUVEC ^ 155 5.4.3 SP treatment does not alter SMC viability following H/R. ^ 158 5.4.4 Effect of SP and COX-inhibition on 8-isoprostane production following H/R in CYP 2C9 expressing HUVECs ^ 158 5.5 Discussion ^ 161 5.6 Bibliography ^ 164 Chapter 6: Summary and Conclusions ^ 166 6.1 Restatement of the Problem ^ 166 6.2 Summary of Findings ^ 167 6.3 Relevance of Findings ^ 170 6.4 Future Directions ^ 171 6.5 Concluding Remarks ^ 172 6.6 Bibliography ^ 174 Appendix I: Animal Care Certificate for Transplantation ^ 176 Appendix II: Rat Heterotopic Heart Transplantation SOP ^ 178 Appendix III: List of Publications, Abstract, Oral Presentation and Awards ^ 186  vi  List of Tables  Table 1.1 2004 Revised ISHLT heart biopsy grading categories for cellular and antibodymediated rejection. ^ 7 Table 4.1 Peripheral cytokine and chemokine levels following heterotopic heart transplantation in SP treated and control rats. ^ 139 Table 4.2 Repeated measures analysis of peripheral cytokine and chemokine levels following heterotopic heart transplantation in SP treated and control rats. ^ 140  vii  List of Figures Figure 1.1 ISHLT Kaplan-Meier survival curves for adult heart transplantation by era ^ 4 Figure 1.2 Histology of CAV ^ 11 Figure 1.3 Pathogenesis of CAV. ^ 12 Figure 1.4 The CYP monooxygenase reaction cycle ^ 28 Figure 1.5 Overview of the three pathways of arachidonic acid metabolism^ 37 Figure 2.1 A Map of the pTAT-HA-fusion protein. ^ 64 Figure 2.2 HCASMCs and HUVECs express ARC. ^ 71 Figure 2.3 TAT-ARC fusion protein transduction into HCASMCs and HUVECs. ^ 72 Figure 2.4 TAT-ARC uptake into HUVECs and HCASMCs is punctate and concentrationdependent. ^ 74 Figure 2.5 TAT-ARC does not confer greater protection against H202 in HUVECs than TAT0-gal control ^ 75 Figure 2.6 TAT-ARC does not confer greater protection against H202 in HCASMCs than TAT-0-gal control ^ 76 Figure 2.7 Overexpression of ARC prevents H202-induced cell death. ^ 78 Figure 2.8 Overexpression of ARC prevents myogenic differentiation. ^ 79 Figure 2.9 Overexpression of ARC prevents myogenic differentiation. ^ 80 Figure 2.10 Overexpression of ARC prevents myogenic differentiation. ^ 81 Figure 2.11 ARC stable overexpression prevents the expression of the muscle-specific markers troponin T and myogenin. ^ 83 Figure 2.12 Transient ARC overexpression prevents the expression of the muscle-specific markers troponin T and myogenin. ^ 84 Figure 2.13 ARC levels increase in H9c2 cells upon differentiation. ^ 85 Figure 2.14 ARC overexpression prevents caspase-3/7 activity during differentiation. ^ 86 Figure 3.1 Sulfaphenazole (SP) restores post-ischemic endothelium-dependent NO-mediated vasodilation. ^ 99 Figure 3.2 SP does not restore post-ischemic endothelium-independent vasodilation produced by sodium nitroprusside (SNP). ^ 101 Figure 3.3 SP does not restore post-ischemic endothelium-independent vasodilation produced by isoproterenol. ^ 102 Figure 3.4 Constrictor responses to KCl were unaffected by SP pre-treatment. ^ 103 Figure 3.5 SP reduces ROS production following UR ^ 104 Figure 3.6 SP reduces ROS production following UR ^ 105 Figure 3.7 Peroxynitrite measurements in post-ischemic coronary effluent. ^ 106 Figure 3.8 Proposed mechanism of CYP 2C induced impaired post-ischemic vasodilation. 109 Figure 4.1 SP treatment does not reduce post-transplant weight gain. 123 Figure 4.2 CYP 2C6 is expressed in transplanted rat heart blood vessels and myocardium.124 Figure 4.3 SP administration at time of surgery attenuates allograft luminal narrowing ^ 125 Figure 4.4 SP administration at time of surgery attenuates allograft luminal narrowing ^ 126 Figure 4.5 Histological features of diffuse and focal myocardial infiltration ^ 127 Figure 4.6 Histological features of epicardial and endocardial immune infiltration. ^ 128 Figure 4.7 CYP 2C does not contribute to general myocardial immune infiltration. ^ 130 Figure 4.8 CYP 2C does not contribute to perivascular immune infiltration. ^ 131 viii  Figure 4.9 Perivascular immune infiltration in the absence of luminal narrowing. ^ 132 Figure 4.10 SP treatment does not alter CD3 + lymphocyte infiltration in the vasculature ^ 133 Figure 4.11 SP treatment does not alter CD8 + lymphocyte infiltration in the vasculature ^ 134 Figure 4.12 CYP 2C does not significantly contribute to post-transplant apoptosis. ^ 135 Figure 4.13 CYP 2C-inhibition has an insignificant effect on EC loss at day 4 post-transplant. ^ 136 Figure 4.13 CYP 2C contributes to SMC proliferation. ^ 138 Figure 4.14 CYP 2C contributes to peripheral IFN-y levels post-transplantation ^ 141 Figure 4.15 CYP 2C contributes to post-transplant serum free 8-isoprostane levels. ^ 143 Figure 5.1 Relationship between hypoxic chamber oxygen concentration and measured P02. ^ 154 Figure 5.2 CYP 2C9 expression in HUVECs following adenoviral transfection and H/R ^ 156 Figure 5.3 CYP 2C9 expression in HUVECs reduces cell viability following H/R ^ 157 Figure 5.4 SP treatment in HCASMCs does not alter proliferation or cell viability following H/R^ 159 Figure 5.5 CYP 2C increases 8-isoprostane levels. ^ 160  ix  List of Symbols, Abbreviations and Acronyms A^AA^Arachidonic acid ACh^Acetylcholine AIDS^Acquired immune deficiency syndrome AM^Acetoxymethyl AMR^Antibody-mediated rejection ARC^Apoptosis repressor with caspase recruitment domain C^[Ca2-1c^Intracellular calcium levels [Ca 21„,^Mitochondrial calcium levels CARD^Caspase recruitment domain CAV^Cardiac allograft vasculopathy CK^Creatine kinase CMV^Cytomegalovirus COX^Cyclooxygenase CYP^Cytochrome p450 D DC^Dendritic cell DEA^Dihydroxyeicosatraenoic acid DHE^Dihydroethidium DMEM^Dulbecco's modified eagle's medium E(B/G)M Endothelial basal/growth medium E EC^Endothelial cell EDHF^Endothelium derived hyperpolarizing factor EET^Epoxyecosotrienoic acid EGF^Endothelial growth factor eNOS^Endothelial nitric oxide synthase ERK^Extracellular signal-regulated kinases F^F344^Fisher 344 rat FBS^Foetal bovine serum FCS^Foetal calf serum GA-1000 Gentomycin-amphotericin B G GM-CSF^Granulocyte macrophage colony-stimulating factor GRO/KC Growth-related oncogene H/R^Hypoxia and re-oxygenation H H2O2^Hydrogen peroxide HAR^Hyperacute rejection HCASMC Human coronary artery smooth muscle cell HE 1E^Hydroxyecosatraenoic acid HLM^Human liver microsomes HPE1E^Hydroperoxyeicosatraenoic acid HS^Horse serum HUVEC^Human umbilical venous endothelial cell I/R^Ischemia and reperfusion  ICAM IFN-y IHC IL iNOS iPLA2 ISHLT IVUS L LOX LT M^MAPK MCP MHC MI MTS Neo N NF-KB NK NO• O 02-• ON00OxLDL P PARP PECAM PG PGI2 PKC PLA2 PVS R RNS ROS RT-PCR S^SERCA Sm(B/G)M SMC SNP SNP SOD SP T^TAT TNF-a TX  Intracellular adhesion marker Interferon gamma Immunohistochemistry Interleukin Inducible nitric oxide synthase Inducible phospholipase A2 International society for heart and lung transplantation Intravascular ultrasound Lipoxygenase Leukotrienes Mitogen activated protein kinase Monocyte chemoattractant protein Major histocompatibility complex Myocardial infarction CellTiter96 AQuoeus assay Neomycin Nuclear factor kappa B Natural killer Nitric oxide Superoxide Peroxynitrite Oxidized low density lipoprotein Poly (ADP-ribose) polymerase Platelet endothelial cell adhesion molecule Prostaglandin Prostacyclin Protein kinase C Phospholipase A2 Perivascular space Reactive nitrogen species Reactive oxygen species Reverse transcriptase - polymerase chain reaction Sarco/endoplamsic reticulum calcium ATPase Smooth muscle basal/growth medium Smooth muscle cell Single nucleotide polymorphism Sodium nitroprusside Superoxide dismutase Sulfaphenazole Transactivator of transcription Tumour necrosis factor alpha Thromboxane  Acknowledgements Firstly, I would like to thank my primary supervisor and mentor, Dr. David Granville. Dave's drive towards discovery, creativity and productivity has encouraged me to set my bar higher and push to obtain my goals. He has also challenged me to think and work independently and to take the lead in my research. As a result, Dave has put me on my way towards becoming an innovative and productive researcher. He has also encouraged me to always keep my eyes open, never to discount unexpected findings and, of course, has imparted his wisdom about the importance of a good lunch. I would also like to thank my cosupervisor Dr. Bruce McManus. Bruce's immense understanding of inflammatory cardiovascular diseases, including cardiac allograft vasculopathy, has been a tremendous asset to this project. In addition, Bruce's fire and determination has been inspirational. Thank-you kindly to my advisory committee: Drs. Issy Laher, Bruce Verchere and, my chair, Wan Lam, for all their suggestions and support. I would like to thank the Heart and Stroke Foundation (H&SF) of BC and Yukon for providing operating funds supporting this research. Thank-you to the H&SF of Canada, The Michael Smith Foundation for Health Research (MSFHR) and The Canadian Institutes for Health Research/MSFHR Transplantation Training Program for personal support in the form of salary awards. I would like the thank Dr. Roberta Gottleib and Dr. Ingrid Fleming for supplying the TAT-ARC Construct and the CYP 2C9 adenoviral constructs, respectively. Thanks also go to Amrit Aitken for assistance with staining and sectioning and Dr. Ryon Bateman for assistance with P02 measurements. A tremendous thanks to all 'the Granvillites': Hongyan  xii  Zhoa, Rani Cruz, Wendy Boivin, Ciara Chamberlain, Lisa Ang and Paul Hiebert; and to all our 'honorary Granvillites'; most notably, Erin Tranfield for their day to day help and for making the laboratory a great place to be everyday. I owe a debt of gratitude to the cooperative education and summer students that I have worked with throughout my studies: Shirley Chen, Kellyann Jones, Eric Venos, Munreet Chehal, Katelyn Mueller and Paul Hiebert. I also owe a special gratitude to the late Dr. Sasha Kerjner whose surgical expertise in heterotopic transplantation was invaluable to this project and whose sense of humour and kindness will ensure her a warm place in my memories. Finally, I would like to thank my family. Thank-you to my parents, Ann and Don Hunter, my sisters, Carly and Sarah, and their husbands, Jon and Beau, and Damon for their love, support and encouragement.  Co-Authorship Statement Chapter 2 is based on the manuscript "Apoptosis repressor with caspase recruitment domain  (ARC) inhibits myogenic differentiation." It was published in FEBS Letters, 2007, volume 581(6), pages 879-84. This manuscript was co-authored with Jingchun Zhang, Shirley C. Chen, Dr. Xiaoning Si, Brian Wong, Dr. Daryoush Ekhterae, Dr. Honglin Luo and Dr. David J. Granville. I worked with the senior authors on this paper to develop the research plan for this paper. I was assisted by Jingchun Zhang and Dr. Xiaoning Si with the adenoviral infection experiments and quantitation of multinucleation. Shirley Chen was a co-operative education student under my supervision and assisted with collection of cell lysates and Western blotting analyses on experiments related to stably transfected cells. Brian Wong provided valuable assistance in experiments related to immunofluorescence staining. Dr. Daryoush Ekhterae provided the stably transfected cell lines for this paper. Drs. Luo and Granville are co-senior authors on this publication. I was primarily responsible for writing the manuscript and assisted the senior authors with the editing and response to reviewers.  Chapter 3 is based on the manuscript "Cytochrome p450 2C inhibition reduces post-  ischemic vascular dysfunction" published in Vascular Pharmacology, 2005, volume 43(4), pages 213-9. This manuscript was co-authored with Dr. Ni Bai, Dr. Ismail Laher and Dr. David Granville. Dr. Ni Bai was co-first author on this paper and was primarily responsible for heart isolation, isolation of septal arteries and artery cannulation. Drs. Laher and Granville are co-senior authors on this publication. I, along with the two senior authors, designed the experiments described in the paper. I was responsible for the majority of writing xiv  of the manuscript. I performed all assays assessing reactive oxygen species production and assisted with assays related to induction of ischemia and reperfusion and measurement of vascular function.  Chapter 4 is based on the manuscript "Cytochrome p450 2C enzymes contribute peii-  transplant ischemic injury and cardiac allograft vasculopathy" in revisions for The American Journal of Transplantation. This manuscript was co-authored with Dr. Alexandra Kerjner,  Katelyn Mueller, Dr. Bruce McManus and Dr. David Granville. Dr. Kerjner performed the surgical aspects of the rat heterotopic cardiac transplantation. Katelyn Mueller was a cooperative student under my supervision who assisted with some of the immunohistochemical studies. Dr. McManus provided insight into the grading of the immune infiltration. Dr. Granville is the senior author on this publication. I developed the experimental approach together with the senior author and helped to write the grant that funded this research. I conducted the bench work and wrote the paper which was reviewed and edited by the senior author.  Chapter 5 is based on a manuscript currently in preparation. Co-authorship is held by me,  Paul Hiebert and Dr. David Granville. Paul Hiebert is a co-operative education student under my supervision that assisted with measurements of arachidonic acid metabolites. Dr. Granville is the senior author on this publication. I, along with the senior author, developed the research plan for this project and carried out the experimental protocols described in this chapter.  xv  Chapter 1  Chapter 1: Introduction  1.1 Cardiac Transplantation 1.1.1 Historical perspective  Although organ transplantation became a viable therapeutic strategy only in the past twenty-five years, the concept of exchanging organs and tissues between individuals has existed for millennia. Early references to organ transplantation include the Chinese physician Pien Ch'iao reportedly exchanged hearts between a man of weak will and a man of strong will in 500 B.C. and in the third century A.D. the Roman Catholic saints Damian and Cosmas reportedly replaced the gangrenous leg of a Roman Deacon with the leg of a recently deceased Ethiopian. 1 While there were many documented, and likely many undocumented, attempts at organ transplantation prior to the 20 111 century, it was not until this time that major advancements in the field of transplantation occurred. This section does not aim to provide a complete history of transplantation but simply to highlight major findings that furthered the advancement of the field and underline those areas for which significant research is needed. The first systemic study of transplantation occurred in 1908 when Alexis Carrel performed double kidney exchanges between cats. This study was made possible due to the development, by Carrel with Charles Guthrie, of the technique of artery and vein anastomoses. 2This technique, still used today, laid the groundwork for solid organ transplantation and many other vascular procedures and won Alexis Carrel the Nobel Prize in Physiology or Medicine in 1912. Although none of the cats in Carrel's study survived, some 1  Chapter 1 were able to maintain urinary output for up to 25 days, thus demonstrating that organ transplantation was viable at the surgical level. The discovery of human ABO blood groups by Karl Landsteiner in 1900 combined with the hypothesis of Peter Medawar that transplant rejection was an immunological process 3 lead to the first successful solid organ transplant; performed in 1954 by Joseph Murray, involving a kidney transplant between identical twins.  4  Unlike other solid organ transplants, cardiac orthotopic transplantation was not surgically viable until the development, in the early 1950s, of the heart-lung bypass machine, credited to John Gibbon. James Hardy attempted the first documented human heart transplant in 1964. Unfortunately the recipient's heart failed prior to a human donor becoming available so Hardy proceeded using a chimpanzee heart which quickly failed due to hyperacute rejection (described in section 1.2.1). 5 In 1967, Christiaan Barnard performed the first successful heart transplant in South Africa with the recipient surviving for eighteen days following transplantation before dying as a result of pneumonia. 6 This lead to over 100 heart transplants being performed in the late 1960s. 7 Unfortunately the results were disappointing with a mean survival of only 29 days and many centres discontinued their cardiac transplantation programs. 3 However, during this time Dr. Norman Shumway, at Stanford University, continued programs in both transplantation research and clinical transplantation. His team developed techniques in simplified orthotopic surgical procedures, organ preservation by hyperthermia and rejection monitoring by electrocardiography and serial biopsy. 3 By the end of the 1970s the Stanford transplantation program had improved their 1 year survival level from 22% to 65%. 8 For his efforts, Dr. Shumway is widely considered the father of modern clinical cardiac transplantation.3  2  Chapter 1 The discovery of potent immunosuppressive drugs was equally as important in the history of transplantation as the aforementioned surgical advances. As early as 1951, Peter Medawar, working for the National Institute for Medical Research, suggested immunosuppressive drugs could be used in transplantation. 9 However, the drugs available at the time; namely, cortisone and azathioprine, were not strong enough immunosuppressors for most types of transplantation. In 1980, a sufficiently potent immunosuppressor was discovered in cyclosporin. 1015 Since that time, many other important immunosuppressive drugs have been used in transplantation including: prednisone, tacrolimus, rapamycin, azothioprine and mycophenolic acid. As of 2006, The International Society for Heart and Lung Transplantation (ISHLT) published that approximately 3,000 heart transplants are performed and reported to the society annually. 16 Of those transplants reported, heart transplant recipients can now expect a 1-year survival rate approaching 90% and an average graft life of 10.3 years (Figure 1.1).  16  Despite these impressive achievements, the field of cardiac transplantation still has a long way to go. Malignancies and infections due to immunosuppressive regimes is currently the largest cause of death amongst transplant recipients and the largest impediments to long term graft survival is chronic heart transplant rejection in the form of cardiac allograft vasculopathy (CAV, described in detail in section 1.1.3). 17 The pathogenesis of CAV is the central focus of this thesis.  3  Chapter 1  100 All comparisons significant at p < 0.0001 80^-•  CO  60 - - - 1982-1991 (N=18,844)  40 — -  — - 1992-2001 (N=34,987)  CO  •  •  2002-6/2005 (N=9,459)  20 HALF LIFE 1982 1991: 8.9 years; 1992-2001: 10.3 years; 2002-6/2005: NA -  -  0 0^1^2 3^4 5^6 7^8 9 10 11 12 13 14 15  Years  Figure 1.1 ISHLT Kaplan-Meier survival curves for adult heart transplantation by era.  Survival of adult heart transplant recipients as calculated using the Kaplan-Meier method as illustrated by the ISHLT registry slides. 17 Graph incorporates information from all transplants where follow-up is available with unavailable data being estimates rather than exact rates. The half-life is defined as the time point at which 50% of all of the recipients have died. Comparisons were made using log-rank test statistic.  4  Chapter 1 1.1.2 Hyperacute and acute forms of cardiac transplant rejection Transplant rejection is the process by which the transplant recipient's immune system recognizes the transplanted organ, tissue or cells as foreign and attempts to destroy it/them. The main types of transplant rejection are hyperacute, acute cellular rejection, acute antibody-mediated rejection and CAV. This section aims to provide a brief overview of the hyperacute and acute forms of allograft rejection. As CAV is central to this thesis, a more detailed discussion of its pathological features and aetiology is provided in section 1.1.3.  1.1.2.1 Hyperacute rejection Hyperacute rejection (HAR), also termed antibody-mediated rejection, describes the process of graft destruction within minutes to hours following transplantation. 18 HAR is caused when the recipient has pre-formed circulating antibodies to endothelial antigens present on the graft; most commonly ABO blood group antigens or major histocompatibility complex (MHC) antigens. 19 The formation of antigen-antibody complexes activates the complement system causing mass neutrophil infiltration, endothelial damage and subsequent micro-thrombi. 19 HAR is now rarely observed in allografts (i.e., grafts between two members of the same species) but can occur if recipients have previously been exposed to MHCantigens present on the graft from prior pregnancies, blood transfusions or transplants or when errors in ABO blood type matching occur. An interesting exception to this happens in infants where ABO blood type matching may not be required. Infants do not produce isohemagglutinins or serum anti-A or anti-B antibodies until 12-14 months of age. 20 Heart transplants have been successfully performed in infants across the ABO barrier without 5  Chapter 1 incident of HAR. 21-23 Unlike with allotransplantation, HAR remains a major hurdle in xenotranplantation (i.e. transplantation between two members of different species) and remains an area of intense research in this field.  1.1.2.2 Acute cellular rejection Acute cellular rejection is a process that can occur starting only days following transplantation but can also occur at any time during the life of the graft. Between 40 and 50% of all transplant recipients will be treated for at least one acute rejection episode within a year of receiving their transplants. 17 Although increasing levels of immunosuppressive drugs are able to combat the majority of acute rejection episodes, over-immunosuppressing patients can lead to malignancies and infections and therefore must be kept in balance. Acute cellular rejection requires alloreactive T-lymphocytes (either CD4 + or CD8 ± ) to recognize alloantigens expressed on the graft resulting in their activation and subsequent proliferation. Immune infiltration then leads to graft cell necrosis and or vessel thrombosis and eventually graft function loss. 24 The endomyocardial biopsy was first described by Caves  et al. in 1973 as a method of monitoring cellular transplant rejection.  25  It remains the 'gold  standard' for monitoring cardiac transplants for signs of rejection. The ISHLT first created a grading scale for histological diagnosis of acute rejection in 1990. 26 This grading scale remained unchanged until it was updated by the Society in 2004. 27 The criteria for the updated grading scale are shown in Table 1.1. Immunosuppressive strategies for rejection vary per transplant centre and often among patients within each centre. Most commonly, triple-drug therapy and cytolytic 6  Chapter 1  Grade code^  Grade criteria  Cellular rejection Grade 0 R Grade 1 R, mild Grade 2 R, moderate Grade 3 R, severe  No rejection Interstitial and/or perivascular infiltrate with up to 1 focus of myocyte damage Two or more foci of infiltrate with associated m oc to damage Diffuse infiltrate with multifocal myocyte damage ± edema, ± hemorrha!e ± vasculitis Antibody-Mediated Rejection (AMR)  AMR 0 AMR 1  Negative for acute AMR No histological or immunopathological features of AMR Positive for AMR Histological feature of AMR Positive immunofluorescence or immunoperoxidase staining for AMR (positive CD68, C4d)  Table 1.1 2004 Revised ISHLT heart biopsy grading categories for cellular and antibody-mediated rejection. Standardized cardiac biopsy grading for acute cellular rejection and acute antibody-mediated rejection as modified from Stewart et al. 27 'R' denotes revised grade to differentiate these grades from the 1990 criteria. 26  7  Chapter 1 therapy are utilized during the peri-operative period. Triple-drug therapy includes prevention of lymphocyte differentiation by interleukin-2 (IL-2) reduction through cyclosporine or tacrolimus, purine synthesis inhibition by azathioprine or mycophenolate mofetil, and lympholytic treatment with corticosteroid therapy. Cytolytic agents employed include OKT3 and ATG/ALG. To reduce the negative impact of the various side effects of each of these treatments doses are decreased following the peri-operative period constituting maintenance immunosuppression. (Reviewed in 28)  1.1.2.3 Antibody-mediated rejection Antibody-mediated rejection (AMR), also termed 'biopsy-negative rejection', `vascular rejection' and 'humoral rejection', is a form of vascular inflammation or damage resulting in hemodynamic compromise where there is minimal evidence of cellular rejection. 29-33 Surprisingly, patients in this category have worse outcomes than patients with higher ISHLT biopsy scores. 34 AMR does not typically respond well to increased immunosuppressive therapy and increases risk of graft loss, CAV and mortality. AMR is characterized by prominent capillaries in the biopsy that have endothelial swelling and deposition of immunoglobulin and complement. 29 Endothelial swelling of the capillaries results from macrophage influx into injured capillaries and thus can be detected using macrophage markers, most commonly CD68. 35 '  36  AMR is typically diagnosed as  present or absent based on the ISHLT guidelines shown in Table 1.1. Current treatment regimes for AMR are limited and usually involve high-dose corticosteroids with more severe cases also requiring cytolytic agents, such as OKT3, and thymoglobulin or gamma globulin, 8  Chapter 1 heparin and antiproliferative agents. 37 Other immunosuppressive drugs including tacrolimus, mycophenolate mofetil and sirolimus have also been used with some success against AMR. 38 ' 39  1.1.3 Chronic cardiac transplant rejection by cardiac allograft vasculopathy (CAV)  CAV is the greatest cause of graft loss for heart transplant recipients surviving 1 year following transplantation. 17 Because transplanted hearts are largely denervated, transplant patients do not experience typical sensations associated with myocardial ischemia or infarction. Therefore, the first clinical indications of CAV can be arrhythmias, congestive heart failure or even sudden death. 4° Using the traditional detection method of angiography, CAV can be detected in up to 42% of patients 5-years post-transplant. 41 Intravascular ultrasound is able to detect much higher levels of CAV with transplant related intimal thickening being detected in 75% of transplant recipients only 1 year following transplantation. 42  1.1.3.1 Pathological characteristics of CAV CAV is a form of arteriosclerosis characterized by diffuse and obliterative intimal thickening. Although CAV is classically described as concentric, fibrous plaques, there are a wide array of abnormal phenotypes including lesions resembling complicated native atherosclerosis. 43 In the latter stages of development allograft arteriosclerosis often involves lipid deposition and calcification. 44 ' 45 Further complicating histological phenotyping is that CAV can occur in regions with existing atheromatous disease. This phenotype is likely to 9  Chapter 1 become more common with transplant programs accepting hearts from donors over 55 years of age, considered marginal donors. CAV involves large and small epicardial and intramural arteries as well as venous structures of the graft. 46  47 '  The recipients native blood vessels are  not affeeted. 4° Both focal plaques and diffuse intimal thickening have been observed in CAV. Contrary to what happens in the intima, the medial layer of the vessels do not thicken and may experience thinning. 48 Figure 1.2 shows the classical phenotype and some of the common pathological characteristics of CAV.  1.1.3.2 Pathobiology of CAV The pathogenesis of CAV, shown in Figure 1.3, is not yet fully elucidated but is believed to involve a chronic allogenic response to the transplanted organ propagated by nonimmunological factors. 49 The importance of the immune system to the development of CAV is evident by the absence of CAV development in isografts. 40 The endothelium of the grafted heart serves as an interface between the allograft and the recipient. Endothelial cells are the first cells to be recognized and are the primary target of the hosts immune system. 49 Nonimmunological factors, as well as, prior episodes of acute and antibody-mediated rejection can contribute to endothelial activation early following transplantation. Although endothelial activation remains a loosely defined term it generally involves increased expression and/or presentation of MHC antigens, adhesion and co-stimulatory molecules, along with altered secretion of cytokines and chemokines. 5° Endothelial activation enhances entry of immune cells, decreases endothelial adhesiveness, contributes to impaired vasomotor function, described in section 1.1.4, and intimal thickening. 51 Both cell-mediated and 10  Chapter 1  Figure 1.2 Histology of CAV. Distal left anterior descending artery from a 16 year old male, 20 months post-transplant from a 47 year old male donor. The artery shows a narrowed lumen (L), a recent, nearly occlusive thrombus (T), a concentrically thickened neointima (NI) and a largely intact media (M). This vessel also has areas of fatty deposits (FD), as well as, areas of collagen (C) and proteoglycan (PG) deposits. The adventitia (A) has multiple enlarged vaso vasora (examples shown with arrows).  1I  Chapter 1 Propagating endothelial Injury  Luminal narrowing  I/R injury Metabolic abnormalities CMV infection Antibody-mediated rejection Cellular rejection damage  _ow  Non-denuding injury  I^MHC & EA expression  Denuding injury  NO Lumen  Endothelium SMC and myofibroblast migration and proliferation  Lipids lntima  Elastic lamena  Figure 1.3 Pathogenesis of CAV. Upon propagating endothelial injury induces both denuding and non-denuding injury of the endothelium. Denuding injury increases endothelial permeability allowing increased entry of T-lymphocytes (T), monocytes and macrophages (M) and lipids further contributing to vascular damage. Non-denuding injury induces MHC expression and presentation of endothelial antigens (EA). Immunogenic endothelium allows antigen expression by dendritic cells (D) and consequent activation and differentiation of T-cells. Non-denuding injury also leads to decreased nitric oxide (NO•) production increasing SMC and myofibroblast migration and proliferation in the neo-intima.  12  Chapter 1 humoral responses have been found to play roles in CAV. Cell-mediated responses primarily involve recognition of MHC antigens through antigen presenting cells or directly by circulating T-lymphocytes. Recipient dendritic cells (DCs) are believed to be the first cell type to recognize foreign MHC molecules on donor endothelial cells. 52 These professional antigen presenting cells can then activate large numbers of T-lymphocytes. Endothelial cells constitutively express both class I and class II MHC antigens and are therefore able to activate CD8 + and CD4 + lymphocytes, respectively, through indirect allorecognition. The degree of MHC antigen mismatch has been found to correlate with the development of CAV. 53 ' 54 During allograft rejection MHC class II is upregulated further enhancing recognition by CD4 + lymphocytes. 55 CD4 + lymphocytes upon activation release IL-2 stimulating the generation of CD8 + cytotoxic lymphocytes, activate alloantibody-producing B cells and macrophages. In a rat heterotopic heart transplant model, depletion of CD4 + but not CD8 + T-cells was able to prevent CAV. 56 Further research into the mechanisms of CD4 ± induced allograft intimal thickening have indicated that a Thl-type response contribute to, 57  58 '  and a Th2-type response may protect against 59 CAV.  Although MHC molecules are important contributors to CAV development, they are not required for its pathogenesis. CAV has been shown in MHC identical grafts and MHC `knockout' animals. 6° The rat heterotopic transplant model, described in section 1.1.5.1, utilized in this thesis does not contain MHC class I or II differences yet the allografts develop CAV. 61 Antibodies against multiple and diverse endothelial antigens have been detected in transplant recipients and have been linked to graft vasculopathy. 62-64 Vimentin has been found as the most prominent antigen in CAV and patients with high levels of anti-vimentin antibodies are at increased risk of graft arteriopathy. 64-66 Antibodies against intracellular 13  Chapter 1 adhesion marker (ICAM)-1 has also been linked to CAV and polymorphisms in ICAM-1 may be protective against rejection. 67 Upon entry of immune cells, the graft vessel wall sustains chronic immune injury. Intimal thickening in CAV is largely due to modified smooth muscle 46 and myofibroblast 68 proliferation, fibrosis and infiltration of macrophage/monocytes and T-lymphocytes.  4°  These  cells are also able to alter cytokine and chemokine levels and augment extracellular matrix synthesis. 24 Endothelial cells in healthy vessels produce nitric oxide (NO•) and antithrombotic proteins. 69 NO• plays a vital role in vascular homeostasis, described in section 1.1.4, but also prevents pro-inflammatory cytokine production by inhibiting nuclear factor KB (NF- KB) and smooth muscle cell (SMC) proliferation. 70-73 CAV causes dysregulation of the NO• synthase pathway leading to impaired NO• production and activity. 51 Endothelial NO• synthase (eNOS) deficient mice have accelerated allograft vasculopathy in aortic transplants. 74 Several cytokines and chemokines are involved in the pathogenesis of CAV. Interferon-gamma (IFN-y) is thought to be a key regulator of graft arteriosclerosis. 75)  (Reviewed in  IFN-y is primarily produced by Thl CD4 + T-cells following induction by IL-12 but can  also be produced by CD8 + T-cells, natural killer (NK) cells, NK T-cells, DC and macrophages. 75 As mentioned above, transplants in mice with a deficient Thl-type response and in IFN-y knockout mice have reduced CAV. 57 ' 58 IFN-y regulates hundreds of genes including pro-inflammatory cytokines and chemokines, growth factors, transcription factors and membrane receptors. 76 IFN-y enhances expression of MHC class I and II molecules 77 ' 78 and induces leukocyte independent SMC proliferation in vascular transplants. 79 Thl CD4 + Tcells also produce IL-2 involved in T-cell expansion. RAN 1ES is an IFN-y-induced 14  Chapter 1 chemokine which increases monocyte adhesion of activated graft endolium. 8° Monocyte chemoattractant protein (MCP)-1, MCP-3 and IL-8 have also been detected in CAV lesions. 5° IL-10 and other Th2-type response cytokines, including IL-4, 5, 6, 9, and 13 appear to be protective against CAV development. 59 Non-immunological factors known to contribute to CAV development include hyperlipidemia, viral infection, immunosuppressive drug toxicity and ischemia and reperfusion (I/R) injury. Hyperlipidemia promotes development of fibrotic intimal hyperplasia and lipid deposition in the native vasculature and, at an accelerated rate, in the vasculature of the allograft. 81 Prospective assessment of simvastatin therapy in transplant recipients has demonstrated that statins increase the survival of transplant patients and decrease the development of CAV by more than 45% 11 years post-transplant. 82 Cytomegalovirus (CMV) is the most common viral infection amongst transplant patients. 5° CMV infection has been linked with CAV development though activation of NF- KB and subsequent production of pro-inflammatory cytokines and SMC proliferation. 83-86 Anti-CMV therapy with CMV hyperimmune globulin and ganciclovir reduced both CMV titres and coronary artery luminal narrowing in heart and heart-lung transplant recipients. 87 Most immunosuppressive drugs have little protective benefit against CAV and their side effects such as hyperlipidemia, glucose intolerance and hypertension may actually contribute to its development. 88 I/R injury and its relationship to CAV development are discussed in detail in section 1.2 and 1.2.2, respectively.  15  Chapter 1 1.1.3.3 Treatment of CAV Treatments to control risk factors, such as CMV infection and hyperlipidemia, may help prevent CAV. Unfortunately, there are relatively few treatments for established CAV. In patients where the CAV is focal and has not spread throughout the vascular tree, percutaneous and surgical interventions may be useful. Percutaneous transluminal coronary angioplasty and coronary artery stenting are used by some transplant programs to extend graft life. 89 Unfortunately, transplant patients have very high levels of restenosis (30-60%) following angioplasty. 90 91 Restenosis levels were lower in a small study of transplant patients that received stents. 92 Coronary artery bypass grafting in patients with CAV carries abnormally high risk of perioperative death with death rates between 33.3% and 40% at various transplant centers. 93-95 For patients with diffuse CAV, retransplantation is often the only option. Among immunosuppressive drugs, sirolimus appears to offer the most protection against CAV development and progression by reducing smooth muscle proliferation and migration, increased NO• production, decreased angiogenesis, and inhibition of fibrosis and extracellular matrix production. 96 Ultimately, preventing the factors that propagate CAV development would offer the greatest advantage to transplant recipients.  1.1.4 Vasomotor function following transplantation Post-transplant abnormalities in endothelium-dependent vasomotion can be detected early following transplantation in both macro and micro vessels. 51 Some of these abnormalities can be indicative of peri-transplant endothelial denudation or dysfunction. Transplanted organs often have partial vasoconstrictory, rather than a vasodilatory, responses 16  Chapter 1 to acetylcholine. 97.  98  This phenomenon may resolve in the months following  transplantation. 99 However many groups have detected impairments in vasodilatory responses to acetylcholine, substance P, exercise, serotonin, and cold-pressor testing both immediately following transplantation and years post-transplant. 100-102 Reduced eNOS expression post-transplant is reported to contribute to impaired acetylcholine-induced vasodilation. 1°3 Endothelium-independent vasodilation is also impaired following heart transplantation. This impairment may be caused by cytotoxic damage to the vascular SMCs leading to medial thinning 104  i mpaired mpa i red SMC contractile responses l°5 .  1.1.4.1 Early vasomotor dysfunction as a predictor of CAV Although some researchers have not found a correlation between early vascular dysfunction and CAV development 99 , other studies have found that early vascular dysfunction is predictive of CAV. Davis et al. 106 found that quantitative angiography measurements of acetylcholine-induced vasodilation correlated with CAV development 1year following transplantation as detected by intravascular ultrasound (NUS). Hollenberg et al. 107 also found a correlation between impaired acetylcholine responses and CAV development detected using angiography. In the latter study, responses to adenosine and nitroglycerine were also assessed but were not found to correlated with future CAV diagnoses. 1°7  17  Chapter 1 1.1.5 Animal models of CAV Animal models for CAV include orthotopic arterial grafting and heterotopic and orthotopic cardiac transplantation. All of these models expose the grafted tissue to the recipient's immune system and have provided valuable insight into CAV's aetiology. The advantages and disadvantages of these models are briefly described below. Orthotopic arterial grafting involves harvesting a section of a major artery, typically the aorta or carotid artery, from a donor and inserting the artery into the identical position in the recipient's arterial system using end-to-end anastomoses.  108  This model has many  advantages, not the least of which is that it is surgically less challenging and can be used in small animal models with lower post-surgical morbidity or mortality (< 2%) 108 than cardiac transplantation. Arterial grafts develop classical features of CAV including intimal thickening, smooth muscle proliferation and immune infiltration. 108 These arteries are maintained at near physiological conditions, unlike with heterotopic transplantation; however, they lack the parenchymal factors that can contribute to rejection. This technique precludes measurements of organ function and associative studies of acute rejection and CAV development. Heterotopic cardiac transplantation involves grafting of a donor heart into a nonphysiological position, most commonly the abdominal cavity, of the recipient. A detailed surgical protocol for abdominal rat heterotopic cardiac transplantation is provided in Appendix I. Similarly to the orthotopic arterial graft model, heterotopic cardiac grafts can be performed in small animal models. This allows researchers to study CAV in inbred and genetically altered populations. This model is advantageous over arterial grafting as it allows  18  Chapter 1 evaluation of acute rejection and graft survival can be monitored easily and non-invasively by abdominal palpation. 1°9 The disadvantage of heterotopic heart transplantation is that the hearts are not physiologically loaded and perfusion of the myocardium by the recipient's circulatory system is retrograde through the ostia and into the coronary circulation. Orthotopic cardiac transplantation parallels human orthotopic heart transplantation and offers the advantages that the hearts are physiologically loaded and that parenchymal effects and acute rejection can also be examined. Unfortunately, the surgical aspects of this procedure are inhibitory in most cases. Specifically, a heart-lung bypass machine is required and thus this operation is generally restricted to canine, swine and other large mammal models. The expense of using large animal models and the lack of genetic and inbred populations makes use of this model rare.  1.1.5.1 Lewis to Fisher 344 rat model of heterotopic heart transplantation We selected the Lewis to Fisher 344 (F344) rat heterotopic heart transplant model for these studies. As mentioned in section 1.1.3.2, Lewis and F344 rats have identical class I and II antigens. 110 Minor histocompatibility differences in the Qua-like RT1.0 locus, as well as, erythrocyte and lymphocyte antigens do differ between the strains. 111 The Lewis-F344 transplant model was originally described as a CAV model in 1993 by Adams et al. 112 This model was ideal for this study as it is a well-established model allowing for comparisons with previous studies. It also utilizes commercially available rat strains, has long-surviving grafts and has a high incidence of graft arteriosclerosis. This model produces CAV lesions in >90% of arteries by 3 weeks post-transplant. 112 These lesions closely resemble human CAV 19  Chapter 1 lesions consisting largely of intimal SMC accumulation. A slight increase in monocellular infiltration and necrosis are observed compared to human CAV. 112  1.2 Ischemia and Reperfusion (UR) Injury  I/R is integral in the pathophysiology of myocardial infarction and is a contributing complication to multiple surgical procedures including cardiac transplantation and coronary bypass. 113-118 I/R results in apoptosis and necrosis in the myocardium. The vascular endothelium is even more susceptible to ischemic damage.  119  I/R is associated with  decreased endothelium-dependent vasodilation, decreased NO levels, increased expression of MHC, adhesion molecules and leukocyte adhesion 120 , and adverse contractile modulatory effects. 121 Ischemia is insufficient or absent blood flow and reperfusion is restoration of blood flow flowing an ischemic period. During ischemia, ATP supplies are depleted leading to an increase in cytosolic calcium ([Ca2+] c ). Elevated [Ca 2+], can be prolonged due to reperfusion injury because of reactive oxygen and nitrogen species (ROS and RNS), such as peroxynitrite (ONOO-) can damage the sarco/endoplasmic reticulum (ER) calcium ATPase (SERCA) inhibiting sequestration of intracellular calcium back into the ER. 122 Elevation of [Ca 2 1 c in turn causes an increase in mitochondria' calcium ([Ca 2±],n) levels associated with increased production of ROS linked to damage of the respiratory chain. 123  20  Chapter 1 1.2.1 Reactive oxygen and nitrogen species in UR ROS and RNS are central players in the pathogenesis of UR. ROS and RNS can be broadly divided into free radicals (1-electron donors) and non-radical oxidants (2-electron donors). Free radical oxidants, such as superoxide (02-) and NOV,• can be highly reactive and can act as both oxidizing and reducing agents as they are capable of both donating and accepting a single electron. Nitric oxide (NO•) is produced by eNOS under basal conditions. Under stressed states NO can also be produced by macrophages and SMCs through inducible NO synthase (iNOS). As described above, NO plays important roles in vascular homeostasis through its induction of endothelium dependent vasodilation, platelet aggregation, and in controlling smooth muscle growth and differentiation. However, when NO- is produced in the presence of 02 - • the two rapidly react in the formation of ONOO-, a non-radical oxidant. 124 Non-radical oxidants are capable of accepting two electrons. Other examples of such oxidants include: hydrogen peroxide (H202), ozone and hypochlorous acid. Non-radical oxidants, like ONOO-, are highly chemically reactive and known to cause cell damage through lipid peroxidation, tyrosine nitration, and reactions with sulfhydryl groups. 125 In addition to its ability to chemically alter many cellular components, ONOO- can also cause cellular dysfunction through the activation of multiple signalling pathways. ONOO- has been shown to increase integrin-dependent adhesion of human neutrophils to human coronary artery endothelial cells through activation of the Raf-1/ extracellular signalregulated kinases (ERK) pathway. 126  It  is also known to activate the ERKs, c-Jun NH2-  terminal kinase, calcium-dependent protein kinase C (PKC), and p38 mitogen activated protein kinase (MAPK). 126 ONOO- can also induce apoptosis. 127-133 Previous research by our centre has shown that acute cardiac rejection and apoptosis is attenuated when mouse cardiac  21  Chapter 1 allografts are transplanted into iNOS knockout recipients compared to iNOS+/+ recipients.  134  Furthermore, 02 - • and ONOO- can disrupt ER calcium ATPases and Ca 2+ regulation in coronary arteries. 122 ' 135-141 ONOO- is also implicated in smooth muscle cell damage through DNA damage and the activation of poly(ADP)ribose synthetase which results in energy depletion. 142 ROS and RNS are generated through multiple pathways including: NAD(P)H oxidase, xanthine oxidase, myeloperoxidase, lipoxygenase, mitochondrial respiration, transition metals, and nitric oxide synthase (NOS). Although they are largely unstudied outside of the hepatic system, cytochrome p450 enzymes (CYPs) can also generate ROS and lead to the production of RNS in cardiovascular tissues. Production of ROS by CYPs is discussed in section 1.4. Antioxidants are capable of significantly preventing or delaying the oxidative damage of substrates which are present at higher concentrations than the antioxidants themselves. Enzymatic antioxidants are perhaps the most well known and are largely responsible for maintaining a reducing intracellular environment in cells. These antioxidants include superoxide dismutases (SODs), catalases and peroxidases.  1.2.2 UR and transplantation  I/R injury plays a significant role in endothelial dysfunction and the pathophysiology  of cAv. 113- 118, 143 The n transplant organ is vulnerable to I/R injury induced by graft ischemia time, quality of graft preservation during transport, hemodynamic status of the donor, catecholamines used for inotropic support, and reperfusion itself. 116 Three sequential phases of graft ischemic time contribute to graft injury during transplantation: (1) the episode of 22  Chapter 1 warm ischemia upon removal of the heart from the donor, (2) the cold ischemic interval associated with storage and preservation of the heart, and (3) the period of warm ischemia during engraftment. 114 Paradoxically, although reperfusion is required to restore tissue oxygenation, much of the damage that ensues during transplantation is associated with the oxidative burst that occurs during reperfusion.  114  Compelling evidence supports a molecular and cellular basis for a causal relationship between UR injury during transplantation and the onset and progression of CAV. 113 ' 143 UR injury to endothelial cells may provide the initial trigger for atherogenesis by stimulating platelet adhesion, release of growth factors, upregulation of MHC Class I and II expression, release of donor antigens, expression of adhesion molecules, and proliferation of vascular smooth muscle cells. (Reviewed in 113-118, 143) Several experimental models using superoxide dismutase and antioxidants have demonstrated the importance of ROS in the pathophysiology of UR injury. However, the development of effective treatments to alleviate reperfusion injury remains elusive. Furthermore, several candidate pathways have been proposed to produce ROS during UR including mitochondria, NADPH oxidases, xanthine oxidase and eNOS. However, the data supporting a role for these systems in UR injury remain inconclusive. For example, targeted deletion of P47Ph07 , an essential component of NADPH oxidase, abrogates NADPHdependent superoxide generation in endothelial cells. However, UR studies in p47-null mice reveal no significant difference in infarct size. 144 Similarly, xanthine oxidase inhibitors have failed to protect against I/R 144 while eNOS may play a protective role.145  23  Chapter 1 Recently, apoptosis repressor with caspase recruitment domain (CARD), described in section 1.3, and cytochrome p450 2C enzymes, described in section 1.4, have been found to play roles in myocardial I/R injury. 146  147 '  The role of these proteins in vascular UR injury  and CAV are unknown.  1.3 Apoptosis Repressor with Caspase Recruitment Domain (ARC)  ARC, apoptosis repressor with caspase recruitment domain, was first identified by Gabriel Nunez's group in 1998. 148 It is a 23 kDa protein with an N-terminal CARD domain and a C-terminal proline/glutamic acid rich domain. Its expression was originally thought to be confined to terminally differentiated skeletal and cardiac muscle. 148 More recently, ARC has been shown to be expressed in cancer cells. 149150 Caspases and other CARD containing proteins are known to bind to one another through this domain. ARC was found to bind and inhibit caspase-2 and 8 indicating important implications in apoptosis. 148 ARC is also able to inhibit potassium efflux associated with apoptosis induction and cell shrinkage.  151  Apoptosis is a tightly controlled form of cell death that is characterized by cell shrinkage, DNA fragmentation and membrane blebbing resulting in the packaging of the cell into membrane-enclosed vesicles. These vesicles are then engulfed by surrounding `professional' (macrophages) or 'non-professional' phagocytes. Endothelial cell (EC) and SMC apoptosis and necrosis have been identified as important factors in the progression of CAV. As described above, numerous factors such as oxidative stress, immune cells and cytokines participate in the induction of cell death in CAV. Therefore multiple deathinducing pathways must be inhibited in order to attenuate vascular damage. ARC is one of 24  Chapter 1 the first known multifactorial apoptosis inhibitors and was shown to inhibit both apoptosis and necrosis in cardiac myoblasts. 152 Therefore, ARC may provide a unique method of inhibiting the multiple apoptotic and necrotic pathways that are triggered in this disease.  1.3.1 ARC in UR injury ARC is protective against ischemic injury and oxidative stress in cardiomyocyte and neuronal cells. Ekhterae et al: 52 were the first to link ARC with protection against oxidative injury. They demonstrated that ARC overexpression was able to protect against hypoxia and re-oxygenation (H/R) induced caspase-3 activation, Poly (ADP-ribose) polymerase (PARP) cleavage and cytochrome c release. 152 Gustafsson et a1. 147 found that human immunodeficiency virus (HIV) transactivator of transcription (TAT)-fusion protein transduction (described in section 2.1) of ARC was protective against oxidative injury induced by H202 in cultured embryonic myocytes and was protective against I/R injury in Langendorff perfused rat hearts. In the latter set of experiments, TAT-ARC transduction reduced both infarct size and creatine kinase (CK) release following I/R. 147 Chatterjee et a/. 153 found similar results using adenoviral transfer of ARC in a rabbit model of regional cardiac ischemia. Treated animals maintained left ventricular geometry, had higher ejection fractions and less border zone fractional shortening that control groups.  153  ARC-deficient  mice demonstrate reduced contractile function, cardiac enlargement, and myocardial fibrosis following aortic banding. 154 These mice also show increased infarct areas following I/R. 154 Studies in hippocampal neurons showed that hypoxia downregulates ARC expression in the hippocampus and that overexpression of ARC protects against hypoxia-induced death in  25  Chapter 1 these cells. 155 There are currently no data indicating whether or not ARC is protective against ischemic injury in the vasculature or whether that protection could reduce CAV development.  1.4 Cytochrome p450 Enzymes (CYPs CYPs are membrane-bound, heme-containing terminal oxidases that are found in organisms ranging from archaebacteria to humans. These enzymes are responsible for the metabolic activation or inactivation of most types of drugs as well as toxins. CYPs oxidize, peroxidize, and/or reduce steroids, arachidonic acid (AA), cholesterol, vitamins and other foreign substances in an oxygen and NADPH-dependent manner. The majority of CYPs isoforms are mono-oxygenases that catalyze the incorporation of a single atom of oxygen into a substrate. CYPs are critical mediators of drug metabolism. Thus, considerable attention has been given to these enzymes by the pharmaceutical industry with respect to their role in drug-drug interactions, drug bioavailability and toxicity.  156  There is substantial inter-individual variation in the activities of various CYP isoforms in humans resulting in differential metabolism, detoxification and/or clearance of xenobiotics. Much of this inter-individual variability can be attributed to polymorphisms in CYP genes resulting in altered activity or expression of the encoded enzyme. However, CYP activity is also heavily influenced by other factors such as drugs, hormones, development, • 157 158 diet and cytolunes. ' Thus, both genetic and epigenetic components determine the ability  of an individual to metabolize a particular drug or toxic substance. To add further complexity, the sequencing of the mouse, rat and human genomes has revealed substantial 26  Chapter 1 differences in the CYP makeup of these animals. 159-16I For instance, the 2J subfamily, which has been shown to be abundantly expressed in the heart, has one member in humans, 4 members in rats and 8 members in mice.  159  Furthermore, mice contain 84 different CYP  isoforms versus only 63 isoforms in humans. 161 Thus, caution must be used when assessing drug metabolism or the activation/deactivation of other toxins in rodents with respect to translating this research to humans as rodents possess CYP isoforms that are not present in humans and vice-versa. This may be one explanation as to why many therapeutics are effective in mice, but fail in humans. The liver expresses the highest levels of CYP and plays a dominant role in the firstpass clearance of ingested xenobiotics and in the regulation of systemic levels of drugs and other chemicals. However, extra-hepatic tissues also possess CYP and contribute not only to first-pass clearance, but may also influence tissue burden of foreign compounds or bioavailability of therapeutics. 162 Many substances require CYP-mediated metabolic activation to form toxicants or carcinogens. The reactive intermediates that are produced are for the most part unstable and unlikely to be transported from the liver to other tissues to exert toxicity. Thus, chemical toxicity in extra-hepatic tissues may be regulated by CYPmediated in situ metabolic activation in the target organ itself. 162  1.4.1 CYP 2C enzymes CYP 2C enzymes are mono-oxygenases that catalyze the transfer of a single oxygen molecule to their substrates. This process requires electron transfer from NADPH to  27  Chapter 1  2H + 3 H2 0 2 02 - + NO• 3 ON0002 +  Figure 1.4 The CYP mono-oxygenase reaction cycle O2 is generated during the CYP reaction cycle when the electrons for the reduction of the central heme iron are transferred on the activated bound oxygen molecule. 0 2 ' is then readily converted to other ROS and RNS through reactions such as those shown for H202 and ONOO-.  28  Chapter 1 cytochrome p450 through electron carriers. 163 This process is shown in Figure 1.4. Unfortunately, this process is relatively inefficient and poorly coupled in eukaryotes, compared to protoplasmic microbial mono-oxygenase systems, leading to the production and release of ROS. 163 CYP mono-oxygenases produce superoxide during three stages of their reaction cycle and can produce superoxide by NADPH consumption even in the absence of substrate. 163 Substrate availability further increases the catalytic activity of CYPs and results in an increase in superoxide production. CYP 2C9 was mapped to chromosome 10 in humans  164 ,  to chromosome 7 in mice  and chromosome 1 in rats. 165 In the heart CYP 2C9 was found to be predominantly expressed on the right side, more specifically in the right ventricle and also in the vasculature. 166 Much of what we know about CYP 2C9 comes from studies related to the metabolism of the many drugs that it metabolizes. Tolbutamide, used in the treatment of type 2 diabetes, is metabolized by CYP 2C9. An uncommon variant of the CYP 2C9 gene seems to be associated with a reduced ability to metabolize tolbutamide. This same variant, CYP 2C9*3 in which isoleucine at position 359 is mutated to leucine, is also associated with both reduced clearance of the anti-inflammatory drug, celecoxib as well as reduced clearance of warfarin, an anticoagulant. 167 X-ray crystallography studies by Williams  et al.  168  elucidated the  structure of CYP 2C9 and discovered a binding pocket in which the anticoagulant, warfarin, binds. Poor metabolizing variants occur at a higher frequency in the white population compared to the black population. 169 Two CYP 2C9 variants have been identified as poor metabolizers of warfarin, CYP 2C9*2 (arg144—>cys) and CYP 2C9*3 described above. In a retrospective cohort study of patients being treated with warfarin, individuals with poor metabolizing CYP 2C9 variants were associated with an increased risk of bleeding events. 17° 29  Chapter 1 Although the rodent equivalent to human CYP 2C9 has not been fully characterized, CYP 2C6 and CYP 2C1 1 are recognized as its putative orthologs. 171 We and others have detected an isoform corresponding to a similar sized protein in rat heart protein extracts using an antibody for human CYP 2C9 and have demonstrated that CYP 2C9 inhibitors reduce post-ischemic superoxide generation in rat hearts.  146' 172  Rat CYP 2C6, but not CYP 2C 11,  has previously been shown to be selectively inhibited by sulfaphenazole in rat liver preparation5. 173  1.4.1.1 CYP 2C in vascular homeostasis Although the majority of CYP are most abundantly expressed in the liver, CYP are also expressed in extra-hepatic tissues including the heart and the vasculature.  174  The human  AA-metabolizing epoxygenases of the 2 gene family; namely 2B, 2C8, 2C9, 2C10, and 2J2, are expressed in the vasculature and have been implicated in vascular homeostasis.  174  These  epoxygenases generate epoxyeicosatrienoic acids (EETs), ROS, and other products. Vascular tone and homeostasis is modulated by numerous vasoactive signals and compounds produced by the autonomic nerves, the tissue, and the endothelium.  174  Vasodilators include vascular flow, the well-known autacoids, NO• and prostacyclin (PGI2), and several less well characterized receptor-mediated agonists. 174 NO/PGI2-independent pathways make a significant contribution to vasodilation, particularly in the renal, mesenteric, and coronary arteries. 174 Endothelium-derived hyperpolarizing factor (EDHF) is an agonist that causes the hyperpolarization of endothelial and smooth muscle cells though both Na-KATPase and calcium-dependent K ± channels.' 75 ' 176 CYP have been linked to EDHF activity 30  Chapter 1 because CYP specific inhibitors, such as 6 (2-proparglyoxyphenyl) hexanamide, can prevent NO/PGI2-independent vasodilation. 177 Furthermore, an antisense approach against the CYP 2C family was able to demonstrate an attenuation of bradykinin-induced EDHF-mediated vascular responses without affecting NO-mediated vascular responses.  178  Moreover,  sulfaphenazole, a selective inhibitor of CYP 2C9, was able to inhibit EDHF-mediated vasodilation in porcine coronary arteries. 179 This research implicates CYP 2C9 as a putative EDHF synthase and 1 1,1 2-EET as the putative EDHF. CYP products such as EETs and hydroecosotraenoic acids (HETEs) as well as their degradation products have been associated with both the induction and inhibition of vasodilation. For example, CYP 2J2 is localized to the endothelium of large and small coronary arteries and is able to generate not only EET from AA, but also epoxyeicosaquatraenoic acids from eicosapentaenoic acid. 18° Both EET and epoxyeicosaquatraenoic acid are known dilators of the microvasculature.  18°  The diol products  of EETs, dihydroxyeicosatrienoic acids, can be taken up by ECs and cardiac myocytes and incorporated into phosphatidylcholine, phosphatidylinositol, and to a lesser extent other phospholipids. 181 Even when EETs are released into the extracellular environment they are believed to incorporate into circulating lipoproteins through esterification. 182 It is hypothesized that the incorporation of EETs into phospholipids serves as a means of storing these molecules, but it is not known if EETs are also active in this form. 174 Unfortunately, the effects of EETs on vasodilation have typically been measured in the presence of inhibitors of NO-dependent vasodilation. This is a concern because NO• is an inhibitor of CYP. For that reason, it is unknown how much of an effect EETs have on vasodilation in the presence of NO•. This is further complicated because CYP also generate ROS during their reaction cycle 31  Chapter 1 as electrons are transferred from the central heme iron to the activated bound oxygen molecule. 179 In fact, CYP make a significant contribution to the cellular production of ROS such as^H202 and hydroxyl radicals. 179 Through the production of free radicals, CYP may also contribute to vascular homeostasis because ROS are known participants in the maintenance of vascular tone and homeostasis. 183 Unlike the EET products of CYP, ROS are implicated in the inhibition of NO-mediated relaxation. 027' reacts with NO• to form ON00 thus reducing the bioavailability and vasoactivity of NO•. 179  1.4.1.2 CYP 2C in YR injury  Yasar et al. (2003) 184 examined correlations between genetic variants of CYP 2C8 and 2C9 and risk of acute MI. An increased risk of acute MI has been associated with the genetic CYP variants CYP 2C9*2 and *3 in female patients, and CYP 2C8*3 in both males and females. 184 These variants have reduced activity compared to their wild-type counterparts. 185, 186 Recent studies from our group suggest that the rat CYP 2C9-equivalent is an important mediator of I/R injury. 146 In the latter study, several CYP inhibitors were tested for their ability to protect against cardiac YR injury. Three structurally-unrelated CYP monooxygenase inhibitors (chloramphenicol; multi-CYP inhibitor, cimetidine; 1A2, 2C6/9, 2D6, 3A4 inhibitor and sulfaphenazole; 2C6/9 inhibitor) were highly protective against I/R injury. The one commonality between these inhibitors was their ability to suppress rat CYP 2C6 or human CYP 2C9. Thus, it became apparent that CYP 2C6/9 may be a key player in cardiac I/R injury. In rat hearts perfused in Langendorff mode, the CYP inhibitors reduced infarct  32  Chapter 1 size, ROS production and CK release compared to that of controls. 146 Similar results were found in a rabbit model of left anterior descending coronary artery constriction. CYP 2C9 inhibitors also increased post-ischemic coronary flow suggesting that increased vasodilation and/or reduced post-ischemic vascular dysfunction plays a role in the cardioprotective effect. The observation that CYP inhibitors attenuate I/R injury is significant. Many risk factors for heart attacks, such as tobacco smoke and cocaine, are potent inducers of CYPs in the heart, while cardioprotective factors, such as resveratrol (found in red wine) and statins, inhibit CYPs. 187-192 Although there are more deaths associated with smoking-induced cardiovascular disease than cancer, the mechanism by which smoking contributes to cardiovascular disease is poorly understood. However, there is evidence to suggest CYP might be responsible. The role of CYP in smoking-related cancer is well-established and recent findings indicate that certain CYP isoforms are involved in atherogenesis. Polymorphisms in CYP 1A1, one of the key detoxifying enzymes catabolizing cigarette smoke-derived toxins, are associated with smoking-induced atherogenesis. CYP 1A1 polymorphisms have been associated with susceptibility to severe coronary artery disease and type 2 diabetes in smokers. 189 Further evidence for a role of CYP in MI stems from studies of cocaine-induced heart attacks. Cocaine can induce acute MI in young adults has been reported to be a potent inducer of CYP in cardiac tissues.  193  190  and  Conversely, although  several mechanisms have been forwarded to explain the cardioprotective effects of polyphenolic compounds found in red wine and other foods 191 ' 194-197 it is of interest to note ,  that these substances are also known CYP inhibitors. 191 ' 194-197 In summary, there is powerful and accumulating indirect evidence supporting a role for CYP in tissue-specific cytotoxicity and cardiovascular disease. 33  Chapter 1  1.4.1.3 CYP 2C in atheromatous disease Thum and Borlak 198 have implicated oxidized low density lipoprotein (oxLDL) in the downregulation of CYP mono-oxygenases in coronary arterial endothelial cells. This study linked oxLDL to increased ROS production and consequent loss in nuclear factor 1 (NF-1) activity. 198 NF-1 is an important regulator of CYP mono-oxygenase expression. 199 A significant decrease in the expression of CYP 1A1, 2A6/7, 2B6/7, 2C8, 2C9, 2E1, and 2J2 was detected in coronary arterial endothelial cells treated with oxLDL, but not in cells treated with normal LDL. 198 Fichtlscherer et al." showed that CYP 2C9 inhibition via sulfaphenazole is associated with increased endothelium-dependent vasodilation in human patients with coronary artery disease. 20° This effect was attributed to a decrease in ROS production by CYPs, as well as a consequent increase in NO• bioavailability and NOmediated vasodilation. This work suggests inhibition of CYP 2C9 as a possible therapeutic intervention to maintain blood flow and protect against ischemic damage in patients with established coronary artery disease."  1.4.1.4 CYP 2C in other cardiovascular diseases CYPs have been speculated to play a significant role, both in the onset of and the protection against a broad spectrum of cardiovascular diseases. While CYPs have been studied extensively in drug metabolism in the liver, studies into their roles in xenobiotic metabolism and the production of biologically active metabolites and toxins in the heart requires further elucidation. CYPs have recently been implicated in the induction of 34  Chapter 1 angiogenesis. Furthermore, endothelial cell proliferation, associated with angiogenesis, is linked with CYP 2C9 expression. Human umbilical vein endothelial cells infected with adenovirus to overexpress CYP 2C9 demonstrated a 50% increase in proliferation over antisense infected cells as well as a 3-fold increase in cyclin D1 expression. 201 This increased endothelial proliferation was prevented with the addition of a CYP 2C9 specific inhibitor sulfaphenazole. 201 Administration of 11,12-EET to chick choriollantoic membranes was able to induce angiogenesis to a similar degree as known pro-angiogenic factors such as endothelial growth factor (EGF) and vascular endothelial growth factor. Again, the induction of angiogenesis by 11,12-EET was inhibited using AG1478 as well as an EGF neutralizing antibody. Similar experiments in a human lung microvascular cell lines showed a more than 25% increase in proliferation with overexpression of CYP 2C9. 202 In this study 14,15-EET, the most abundant EET product of CYP 2C9, was applied to a Matrigel and infused subcutaneously on the dorsal midline of a rat. Angiogenesis was subsequently measured as indicated by haemoglobin content and by immunostaining of platelet endothelial cell adhesion molecule-1 (PECAM). After one week 14,15-EET treated Matrigel showed a 1.6fold increase in haemoglobin over control as well as positive PECAM staining.  202  The  relative contributions of 11,12- and 14,15-EET and the mechanism of EGF receptor involvement in CYP 2C9 induced angiogenesis are currently unknown and require further experimentation to be fully elucidated. CYP 4A1 was also shown to induce angiogenesis in renal interlobar arteries in a smooth-muscle cell dependent manner.  203  Hypertension, or high blood pressure, is a leading cause of death, MI, stroke, and other illness in North America. It is typically asymptomatic and the great majority of patients have essential hypertension, in which the cause of blood pressure elevation is 35  Chapter 1 unknown. 2°4 Hypertension is largely regulated by the cardiac output of the heart, the systemic resistance controlled by blood vessel tone, and the intravascular tone regulated by the kidneys. CYPs have often been considered when treating hypertensive patients due to their interactions with anti-hypertensive drugs such as candesartan  205  , warfarin 206 , phenytoin, and  tolbutamide 207 . Recently, due to the ability of CYPs and their metabolites, EETs and HElEs, to modulate vascular tone and alter renal blood vessels, as described above, they have been linked to the development of hypertension. There have been several reports, however, describing conflicting roles for CYP in hypertension. Fenofibrate, a drug known to induce expression of CYP 4A and elevate production of 20-HE1E, has been shown to reduce blood pressure in stroke prone spontaneously hypertensive rats. 208 On the other hand, the use of 17-octadecynoic acid, an inhibitor of EET and 20-HE'1E production, was also able to reduce blood pressure in Lyon hypertensive rats. 209 Single nucleotide polymorphisms (SNP) of CYP 2C9 were studied to determine if it was possible to predict the efficacy of treatment with irbesartan, a drug used to treat hypertension, with SNP information. 210 The results indicated that the rate of irbesartan metabolism is indicative of the CYP 2C9 genotype expressed, providing a valuable use for genotyping before treatment of hypertension.  1.5 Arachidonic Acid (AA) Metabolism AA is metabolized by three major pathways; the cyclooxygenase (COX) pathway, the lipoxygenase (LOX) pathway, and the CYP epoxygenase pathway. These pathways are shown in Figure 1.5. 36  Chapter 1  Arachidonic Acid  Cytochrome p450 Epoxygenases Lipoxygenases  Cyclooxygenases  DHETEs, HETEs, EETs, ROS  Leukotriene, HPETE, DEA  Prostaglandins, Prostacyclin, Thromboxane A2  Figure 1.5: Overview of the three pathways of arachidonic acid metabolism. AA is metabolized by three main pathways; the cyclooxygenase pathway, the lipoxygenase pathway and the cytochrome p450 epoxygenase pathway. The main products of these pathways are shown as described in 211 212 '  .  37  Chapter 1 1.5.1 AA metabolism by cyclooxygenase The COX pathway results in the cyclization and oxidation of AA, hence its name.  Alternatively, the COX enzymes are known as prostaglandin (PG) H synthases because they lead to formation of PGH. There are three types of COX. COX-1 and COX-3 are constitutively expressed and are present in the stomach, kidney and thrombocytes and the brain, respectively. COX-2 is the inducible form of the enzyme and is present in multiple tissues including the heart and vasculature. However, COX-2 is thought to be constitutively expressed in some tissues including gastric tissues and endothelial cells. 213 Initially COX oxidizes AA into the endoperoxides PGH2 and PGB2 which are precursors of the prostaglandins PGE2, PGF2, PGI2 and of thromboxanes (TX). 213 The production of these metabolites differs in different tissues with TX formation dominating in blood platelets 214 and prostaglandin and prostacyclin formation dominating in vascular cells.  1.5.2 AA metabolism by lipoxygenase  The LOX are dioxygenases that metabolize AA into HPETEs (hydroperoxyeicosatraenoic acids) and DEA (dihydroxyeicosatraenoic acid). These products are then converted to HETEs, leukotrienes, and lipoxins by peroxidases, hydrase and glutathione S-transferase, and lipoxygenases, respectively. There are two main LOX enzymes 5-LOX and 12-LOX, defined by the carbon atom on which the oxygen is fixed. 5LOX is present in many cell types and leads to the formation of leukotrienes (LT) A4, B4 and subsequently through modifications LTC4, LTD4 and LTE4. 12-LOX is more restricted in its expression being present in skin, thrombocytes and some tumours. 38  Chapter 1 1.5.3 AA metabolism by CYPs  The epoxygenase pathway employs CYP epoxygenases in the formation of EETs and HETEs. 211 CYP 2C are the primary epoxygenases involved in AA metabolism by the third pathway. 215 Unfortunately, in addition to the production of EETs and HETEs, CYP 2C also make a significant contribution to the cellular production of ROS such as 02', H202 and hydroxyl radicals. 179 ' 216 AA metabolism is increased during myocardial ischemia and to an even greater degree during reperfusion. 212 The COX and CYP pathways are largely responsible for the increase in AA metabolism during I/R. This increase is the result of an increase in intracellular calcium levels during FR which activates phospholipase A2 (PLA2). PLA2 catalyses the hydrolysis of AA from membrane phospholipids thus increasing the concentration of free AA in the cytosol. 217-220 AA metabolism, on the whole, is detrimental during UR. Several studies, in multiple cell types, have demonstrated that inhibition of AA metabolism during I/R by inhibition of PLA2 or inducible PLA2 (iPLA2) is cardioprotective. 32-35, 219, 221, 222  AA has been shown to induce oxidative stress in multiple cell types 223-226 and  AA causes CYP-mediated superoxide production in isolated renal microsomes. 216 We have previously demonstrated that CYP 2C contributes to vascular and cardiac post-ischemic 02 - * production, 146, 227 likely as a result of increased AA metabolism.  39  Chapter 1 1.5.4 AA metabolites and cardiovascular disease  COX-2 is constitutively expressed endothelial cells, however, COX-2 levels are also known to be induced by cytokines, growth factors, lipopolysaccharides, prostanoids and substrates. 213 COX-2 is also known to be bound to PGI2 synthase in endothelial cells resulting in PGI2 being the predominant AA metabolite in these cells. PGI2 is anti-thrombotic and vasodilatory and plays a central role in vascular homeostasis. 228 Coxibs, selective COX-2 inhibitors, have been associated with increased cardiovascular events. The first studies were related to rofecoxib, Vioxx, and eventually led to its withdrawal from the market. The VIGOR study, of rofecoxib, showed a nearly 5-fold increased risk of myocardial infarction in those patients that received rofecoxib 229 and a correlation between myocardial infarction and rofecoxib was also found by the APPROVe (Adenomatous Polyp PRevention On Vioxx) study of rofecoxib. 38 Studies related to celecoxib and paracoxib/valdecoxib have also shown an association with increased cardiovascular risks. 23°-232 These later studies resulted in warnings related to increased cardiovascular risks for patients taking celecoxib and the voluntary withdrawal or paracoxib/valdecoxib from the market. Although coxibs have been associated with increased risk of cardiovascular events, and it is likely that decreased PGI2 synthesis contributes to these events, the underlying mechanisms and other contributing factors have not been fully examined. During ischemia increased intracellular calcium levels induces the activation of PLA2 and the subsequent hydrolysis of AA from membrane phospholipids.  217-220  Several AA  metabolites, including COX-2 derived PGI2, are known to be cardioprotective. The negative 40  Chapter 1 cardiovascular effects of COX-2 inhibitors have been largely attributed to decreased prostacyclin production. However, the overall metabolism of AA during I/R has been implicated as a key contributor in the progression of ischemic injury. In support of this notion, inhibition of PLA2 or iPLA2 protects against I/R. 219, 221, 222 Therefore, blocking prostacyclin production upstream of COX-2 at the point of AA hydrolysis does not have the same negative cardiovascular effects. This implies that the effects of COX-2 on the cardiovascular system are more complex than inhibition of PGI2 production alone. CYPs, primarily the CYP 2C epoxygenases, are often referred to as the third pathway of AA metabolism (LOX and COX being the other two). We have previously demonstrated that CYP 2C contributes to vascular dysfunction and myocardial injury following I/R. In the latter study, inhibition of CYP 2C by sulfaphenazole reduced myocardial infarction by nearly 60%. CYP 2C undergoes substrate-induced activation. As AA can be metabolized by one of 3 possible mechanisms, it is logical that if one of these pathways were blocked, that this may result in a shift towards the other 2 pathways and increased activity of these pathways. However, the effect of COX inhibition on AA metabolism by LOX and CYP epoxygenases has not been examined.  1.6 Thesis objectives and hypotheses The overall objective of this thesis is to examine potential mechanisms to reduce peritransplant ischemic injury in the vasculature and to assess the relationship between this form of injury and the development of CAV.  41  Chapter 1 To this end we examined the potential of the anti-apoptotic protein ARC to prevent oxidant cell death in the vasculature. We hypothesized that inhibition of apoptotic and necrotic cell death in the donor heart through increased ARC protein levels would attenuate I/R and thus immune-mediated cell death and chronic transplant rejection caused by CAV.  Results from these experiments are described in Chapter 2. We also examined the contribution of the CYP 2C enzymes to peri-transplant ischemic injury and CAV development. We hypothesized that CYP 2C enzymes play a key role in the pathogenesis of CAV through the production of reactive oxygen species that contribute to inflammation, endothelial damage and dysfunction. Our initial examinations, described in Chapter 3, determined the influence of the rodent CYP 2C9-equivalent on I/Rmediated vascular dysfunction in coronary arteries isolated from Langendorff-perfused hearts. We then assessed the contribution of rodent CYP 2C on peri-transplant ischemic injury and CAV using a rat heterotopic heart transplant model of chronic rejection. Results from these studies are described in Chapter 4. Finally, we assessed the effects of CYP 2C9 on hypoxia/re-oxygenation (H/R) induced cell death in EC and SMC and examined questions related to altered oxidative stress and AA metabolism following CYP 2C9 inhibition. Results described in this thesis demonstrate that ARC does not have similar protective effects against oxidant induced injury in vascular cells as it does in myocytes. Further we serendipitously discovered a novel role for ARC in myogenic differentiation. We have demonstrated that CYP 2C contributes to endothelium-dependent vascular dysfunction and vascular ROS generation following I/R. Inhibition of CYP 2C during cardiac transplantation was found to be protective against CAV development and that expression of CYP 2C9 increases cell death and may alter AA metabolism in cultured human ECs. 42  Chapter 1  1.7 Bibliography  1.  Squifflet JP. From leg transplantation by St Cosmas and St Damian to the modern era. Acta Chir Belg. May-Jun 2003;103(3 Spec No):6-9. 2. Carrel A. Landmark article, Nov 14, 1908: Results of the transplantation of blood vessels, organs and limbs. By Alexis Carrel. JAMA. Aug 19 1983;250(7):944-953. 3. Kirklin JK YJ, McGiffin DC. Cardiac Transplantation. 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Jul-Aug 2005;42(4):312-324. 214. Helliwell RJ, Adams LF, Mitchell MD. Prostaglandin synthases: recent developments and a novel hypothesis. Prostaglandins Leukot Essent Fatty Acids. Feb 2004;70(2):101-113. 215. Luo G, Zeldin DC, Blaisdell JA, et al. Cloning and expression of murine CYP2Cs and their ability to metabolize arachidonic acid. Arch Biochem Biophys. Sep 1 1998;357(1):45-57. 216. Fulton D, McGiff JC, Wolin MS, et al. Evidence against a cytochrome P450-derived reactive oxygen species as the mediator of the nitric oxide-independent vasodilator 55  Chapter 1  217. 218. 219. 220. 221. 222. 223. 224.  225.  226. 227. 228. 229. 230.  effect of bradykinin in the perfused heart of the rat. J Pharmacol Exp Ther. Feb 1997;280(2):702-709. Freyss-Beguin M, Millanvoye-van Brussel E, Duval D. Effect of oxygen deprivation on metabolism of arachidonic acid by cultures of rat heart cells. Am J Physiol. Aug 1989;257(2 Pt 2):H444-451. Leong LL, Sturm MJ, Ismail Y, et al. Plasma phospholipase A2 activity in clinical acute myocardial infarction. Clin Exp Pharmacol Physiol. Feb 1992 ;19(2): 113-118. Van der Vusse GJ, Reneman RS, van Bilsen M. Accumulation of arachidonic acid in ischemic/reperfused cardiac tissue: possible causes and consequences. Prostaglandins Leukot Essent Fatty Acids. Jul 1997;57(1):85-93. Williams SD, Gottlieb RA. Inhibition of mitochondrial calcium-independent phospholipase A2 (iPLA2) attenuates mitochondrial phospholipid loss and is cardioprotective. Biochem J. Feb 15 2002;362(Pt 1):23-32. Ogata K, Jin MB, Taniguchi M, et al. Attenuation of ischemia and reperfusion injury of canine livers by inhibition of type II phospholipase A2 with LY329722. Transplantation. Apr 27 2001;71(8):1040-1046. Sargent CA, Vesterqvist 0, McCullough JR, et al. Effect of the phospholipase A2 inhibitors quinacrine and 7,7-dimethyleicosadienoic acid in isolated globally ischemic rat hearts. J Pharmacol Exp Ther. Sep 1992;262(3):1161-1167. Czerniecki BJ, Witz G. Arachidonic acid potentiates superoxide anion radical production by murine peritoneal macrophages stimulated with tumor promoters. Carcinogenesis. Oct 1989 ; 10(10): 1769-1775. Mayer AM, Brenic S, Stocker R, et al. Modulation of superoxide generation in in vivo lipopolysaccharide-primed rat alveolar macrophages by arachidonic acid and inhibitors of protein kinase C, phospholipase A2, protein serine-threonine phosphatase(s), protein tyrosine kinase(s) and phosphatase(s). J Pharmacol Exp Ther. Jul 1995;274(1):427-436. Mayer AM, Spitzer JA. Modulation of superoxide generation in in vivo lipopolysaccharide-primed Kupffer cells by staurosporine, okadaic acid, manoalide, arachidonic acid, genistein and sodium orthovanadate. J Pharmacol Exp Ther. Jan 1994;268(1):238-247. Toborek M, Malecki A, Garrido R, et al. Arachidonic acid-induced oxidative injury to cultured spinal cord neurons. J Neurochem. Aug 1999;73(2):684-692. Hunter AL, Bai N, Laher I, et al. Cytochrome p450 2C inhibition reduces postischemic vascular dysfunction. Vascul Pharmacol. Oct 2005;43(4):213-219. Liou JY, Shyue SK, Tsai MJ, et al. Colocalization of prostacyclin synthase with prostaglandin H synthase-1 (PGHS-1) but not phorbol ester-induced PGHS-2 in cultured endothelial cells. J Biol Chem. May 19 2000;275(20):15314-15320. Bombardier C, Laine L, Reicin A, et al. Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. VIGOR Study Group. N Engl J Med. Nov 23 2000;343(21):1520-1528, 1522 p following 1528. Nussmeier NA, Whelton AA, Brown MT, et al. Complications of the COX-2 inhibitors parecoxib and valdecoxib after cardiac surgery. N Engl J Med. Mar 17 2005;352(11):1081-1091.  56  Chapter 1 231. 232.  Ott E, Nussmeier NA, Duke PC, et al. Efficacy and safety of the cyclooxygenase 2 inhibitors parecoxib and valdecoxib in patients undergoing coronary artery bypass surgery. J Thorac Cardiovasc Surg. Jun 2003;125(6):1481-1492. Solomon SD, McMurray JJ, Pfeffer MA, et al. Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma prevention. N Engl J Med. Mar 17 2005;352(11):1071-1080.  57  Chapter 2: Apoptosis Repressor with Caspase Recruitment Domain in Vascular Cell Death and Myogenic Differentiation )  2.1 Introduction Apoptosis is a tightly controlled form of cell death that is characterized by cell shrinkage, DNA fragmentation and membrane blebbing resulting in the packaging of the cell into membrane-enclosed vesicles. These vesicles are then engulfed by surrounding `professional' (macrophages) or 'non-professionar phagocytes. Endothelial cell and smooth muscle cell apoptosis and necrosis have been identified as important factors in the progression of ischemic injury and CAV. Numerous factors such as oxidative stress, immune cells and cytokines participate in the induction of cell death in CAV as reviewed in section 1.1.3.2. Therefore, multiple death inducing pathways must be inhibited in order to attenuate vascular damage. ARC was originally discovered as a caspase-2 and -8-interacting, anti-apoptotic protein that is expressed primarily in the heart and skeletal muscle. 1 ARC is capable of preventing both apoptotic and necrotic cell death by preserving mitochondrial function. 2 More recently, work by Nam et al. 3 has suggested that ARC is a unique protein that is capable of intersecting with both the intrinsic and extrinsic apoptotic pathways. Overexpression of ARC inhibits ischemia-induced apoptosis in cardiomyoblast H9c2 cells by preventing 58 A version of this manuscript has been published. Hunter AL, Zhang J, Chen SC, Si X, Wong B, Ekhterae D, McManus BM, Luo H, Granville DJ. (2007). Prevention of myocyte differentiation by apoptosis repressor with caspase recruitment domain (ARC). FEBS Lett. 581(5):879-84.  Chapter 2 mitochondrial cytochrome c release 4 and, in Langendorff-perfused rat hearts, TATtransduction of ARC was shown to significantly reduce infarct size following ischemia and reperfusion. 5 ARCs ability to inhibit both apoptotic and necrotic forms of cell death when transfected into cardiac myoblasts 4 , may provide a unique method of inhibiting the multiple apoptotic and necrotic pathways involved in ischemic injury and CAV. However, little is known about the expression or activity of ARC in EC or SMCs. HIV TAT-mediated protein transduction has been developed as a highly efficient method of transducing biologically active proteins into cells and tissues in vivo. The technology requires the synthesis of a fusion protein, linking the arginine-rich, 11 amino acid TAT protein transduction domain, to the protein of interest using a bacterial expression vector followed by purification of this fusion protein under soluble or denaturing conditions. TAT fusion proteins can be added directly to cells in culture or injected in vivo into mice. TAT-mediated transduction of IP injected TAT-beta galactosidase ((3-gal) has previously been shown to be detectable and functional in all tissues, including the heart. 6 Protein transduction occurs with nearly equivalent concentrations in all cells in the transduced population within 15 min, in a dose-dependent manner. 7-9  2.2 Aim As ARC has previously been shown to protect against cardiac I/R injury 5 and UR injury is associated with the development of cardiac allograft vasculopathy (discussed in section 1.2.2), we hypothesized that ARC may be protective against peri-transplant ischemic 59  Chapter 2 injury and prevent the development of cardiac allograft vasculopathy. The aim of this chapter is to explore the potential for ARC to protect against oxidative damage in cardiovascular cell types in culture as a marker of ischemic injury. In this study, we examined the native expression of ARC in EC, SMC and cardiomyocytes. We examined the effects of altered ARC levels on protection against oxidative damage. In the course of these experiments we have found compelling evidence that ARC inhibits myoblast differentiation. We examined alterations in native ARC expression following the induction of differentiation as well as the effect of ARC overexpression on muscle cell differentiation using H9c2 rat myoblasts as a model. We demonstrate that ARC expression is increased in differentiated cells and we show, for the first time, that ARC overexpression prevents myoblast differentiation. Taken together, these results provide evidence of a novel bi-functional role for the apoptosis regulatory protein ARC in myoblast differentiation  2.3 Materials and Methods 2.3.1 Cell culture  Pooled human umbilical venous endothelial cells (HUVECs) and human coronary artery smooth muscle cells (HCASMCs) were obtained from Cambrex (Baltimore, MD). HUVECs were cultured in complete endothelial growth medium (EGM: endothelial basal medium supplemented with 0.4% bovine brain extract, 0.1% human endothelial growth factor (hEGF), 0.1% hydrocortisone and 0.1% gentomycin-amphotericin B (GA-1000); Cambrex) plus 5% foetal bovine serum (FBS, Invitrogen). HCASMCs were cultured in complete smooth muscle growth medium (SmGM: smooth muscle basal medium 60  Chapter 2 supplemented with 0.1% insulin, 0.5% human foetal growth factor B, 0.1% GA-1000 and 0.1% hEGF; Cambrex) plus 5% FBS. The rat embryonic cardiac cell line H9c2 was obtained from the American Type Culture Collection (Manassas, VA). They were grown and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum (FCS) up to passage 23. All cells were cultured using sterile technique.  2.3.2 RNA extraction and reverse transcriptase (RT)-PCR RNA was extracted from cultured HUVEC and HCASMCs to assess native ARC expression. A minimum of 2.5 x 10 6 cells were trypsinized and collected and total RNA was extracted using the Qiagen RNeasy kit as per manufacturer's instructions (Qiagen). Briefly, cells were lysed in RLT buffer, homogenized using the Quashredder spin column, and 1 volume of 70% ethanol was added. Samples were then centrifuged through the RNeasy mini column for 15 s at 10,000 rpm. The column was washed once with buffer RW1 and twice with buffer RPE. Samples were eluted in 50 pi of RNase free water (Qiagen). Purified total RNA was then assessed for ARC and 18S (control) expression by RTPCR. Purified RNA was subjected to DNase treatment to remove contaminating genomic DNA. Five micrograms of RNA was combined with 1X DNase I buffer, 5 mM MgC12, 1 mM dNTPs, 1X RNase inhibitor and DNase I. Samples were run at 37°C for 45 min, 99°C for 7 min and then cooled to 4°C. Following DNase treatment, samples were treated with Qiagen pre-mixed random primer RT reaction at 25°C for 10 min, 42°C for 50 min, 95°C for 5 min, and then cooled to 4°C. PCR reactions were carried out under the following conditions: 1X PCR buffer, 1 mM MgC12, 2.5 U/100µ1 Taq, 200 dNTPs, 1.0 ILiM primers. Primer 61  Chapter 2 sequences were as follows: 18s forward (5'-GTAACCCGITGAACCCCATT-3'), reverse (5'-CCATCCAATCGGTAGTAGCG-3'), ARC forward (5'GGAAACGCCTGGTCGAGAC-3') and reverse (5'-GCTTCAGCCTCGGGTTCC-3'). Thermocycler conditions utilized involved 30 cycles at 94°C, 52°C, 72°C for 1 min each. Products were separated by electrophoresis for 30 min at 100 V through a 1% agarose gel pre-stained with ethidium bromide. Gels were imaged using the Strategene EagleEye II ultraviolet imager (Stratagene, La Jolla, CA).  23.3 Cell lysis and Western blotting  Cells were washed two times with ice cold PBS and lysed in CellLytic M lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich, Oakville, ON). Protein concentrations were measured using the Bio-Rad protein assay which is a modified Bradford protein assay (Bio-Rad, Hercules, CA). This assay measures the change in absorbance of Coomassie Brilliant Blue G-250 to 595 nm upon binding to basic and aromatic amino acids in proteins. Equal amounts of protein were separated by sodium dodecyl sulphate — polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. After blocking with 5% skim milk, the membranes were incubated for 1 h with primary antibodies (1:1000 antimyogenin antibody and 1:200 anti-skeleton muscle troponin T antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or 1:1000 anti-ARC antibody (Alexis Co., Lausen, Switzerland)), followed by incubation for 1 h with 1:4000 IRDye800 Tm or 1:2000 IRDye700 Tm—conjugated secondary antibodies (Rockland Inc. Gilbertsville, PA). Protein expression was detected by using the Odyssey Infrared Imaging System from LI-COR Biosciences (Lincoln, NE). 62  Chapter 2 2.3.4 TAT protein expression, purification and Texas red staining BL21(DE3)pLysS bacteria containing either the pTAT-HA-hARC plasmid or the pTAT-HA-P-gal plasmid were prepared as previously described 5 and kindly provided by Dr. Roberta Gottlieb. The plasmids are modified from the pTAT-HA vector originally developed by Dowdy's group. 1° A map of this plasmid based on the pRSET vector (Invitrogen, Burlington, ON) is shown in Figure 2.1. It contains elements for ampicillin resistance, a T7 promoter, 6x-Histadine (His) and hemagglutinin (HA) tags, and an Nterminal TAT peptide fusion cassette. Frozen glycerol stock cultures were transferred to Luria broth (LB) containing ampicillin (50 µg/ml) to select for transformed bacteria and isopropylthiogalactoside (IPTG, 100 04) to induce expression and were cultured overnight at 37°C with shaking. Bacteria were centrifuged at 5000 rpm for 15 min, washed once with PBS, and resuspended in Buffer Z (8 M Urea, 100 mM NaCl, 20 mM HEPES pH 8.0) containing 20 mM imidazole. Bacterial solutions were then sonicated on ice 3 times for 15 s pulses with 30 s on ice in between pulses and then centrifuged at 11700 rpm for 30 min. Supernatants were collected. Nickle-nitrilotriacetic acid (Ni-NTA) absorbent columns were used to purify the 6xHis tagged proteins. Ni-NTA is a tetradentate chelating adsorbent which occupies four of the six ligand binding sites in the coordination sphere of the nickel ion, leaving two sites free to interact with the 6xHis tag. The NTA is able to stably bind metal ions and retain them under stringent wash conditions. The theoretical capability of this technique allows purification of proteins from less than 1% of the total protein preparation to more than 95% homogeneity in just one step. 11 Ni-NTA columns (5 ml, Qiagen, Mississauga, ON) were 63  ^  Chapter 2  0^° 0. CS) _C^o — z ct^co in cc -  YGRKKRRQRR  N  T 7 ./  -  RBS-ATG-His 6—TAT— HA- MCS  term /  pRSET A,B.0 2.9 kb  Figure 2.1 A Map of the pTAT-HA-fusion protein.  The pTAT-HA-fusion plasmid developed by Nagahara et al. based on the pRSET plasmid. 1° Upon insertion of the desired gene sequence into the multiple cloning site (MCS) the plasmid produces a 6xHis, HA tagged TAT-fusion protein.  64  Chapter 2 prepared by adding 10 ml of resuspended Ni-NTA resin and allowing excess fluid to run through by gravity. Columns were then pre-equilibrated with 50 ml of Buffer Z + 20 mM imidazole followed by the supernatants prepared above. Columns were then washed with 10 bed volumes (2 x 25 ml) of Buffer Z + 10 mM imidazole. TAT-fusion proteins were then eluted by adding 10 ml of 250 mM imidazole in Buffer Z. Elution fractions were analyzed by SDS-PAGE and Western blot. Elution fractions containing TAT-fusion proteins were then desalted and concentrated. PD-10 desalting columns, purchased from Amersham Pharmacia (Piscataway, NJ), were drained and equilibrated with 25 ml of sterile PBS. Elution fractions were then added and samples were eluted in PBS. PD-10 elution fractions were again analyzed via SDS-PAGE, samples containing the highest protein levels were pooled and protein concentration was measured using the Bio-Rad modified Bradford assay described in section 2.2.3. To assess subcellular localization some TAT-fusion protein preparations were stained with Texas red succinimidyl ester (Molecular Probes, Eugene, OR). Texas red (12 mM in DMSO) was added at a molar ratio of dye to protein of 5:1 in 0.1 M bicarbonate buffer (pH 8.3). The mixture was incubated for 60 min at 4°C followed by 30 min at room temperature. These conditions allow amide bonds to form with protein amines but prevent hydrolysis of the dye. Protein-dye mixtures were then desalted using PD-10 columns as described above.  2.3.5 TAT-fusion protein transduction and detection Cells were grown to 70-90% confluency in complete media with 5% FBS. Media was removed and cells were washed with Dulbecco's PBS (DPBS). Media was then replaced with 65  Chapter 2 serum free basal media. TAT-fusion proteins were added between 0-1 1,IM and were incubated for 1 h at 37°C. Cells were then washed twice with DPBS and were then utilized for further experimentation. To assess levels of TAT-fusion protein transduction we utilized proteins pre-stained with Texas Red. Following transduction media was replaced with phenol red free media and cells were imaged using the fluorescent microscope (595-605 nm excitation, 615 nm emission).  2.3.6 Cell viability  HUVECs and HCASMCs were seeded in 6-well plates, grown to 70-90% confluency, and were treated with 0, 62.5, 125, 250 or 500 nM of either TAT-ARC or TAT-13-gal as described in section 2.2.5. Cells were then treated with 0.6 mM H202 for 4 h. Viability was assessed using the CellTiter96TM AQueous Assay (MTS) (Promega, Madison, WI). MTS is a colorimetric assay involving a cell permeable novel tetrazolium compound [344,5dimethylthiazol-2-y1)-5-(3-carboxymethoxypheny1)-2-(4-sulfopheny1)-2H-tetrazolium] and an electron coupling reagent (phenazine methosulfate) PMS. Upon entry in to viable cells, MTS is bioreduced to an aqueous soluble formazan product by dehydrogenase enzymes. The soluble formazan product is proportional to the number of viable cells and can be measured spectrophoretically due to its absorbance at 490 nm. MTS was protected from light and was added at a 1:5 ratio of MTS: media and the reaction was allowed to proceed for 1 h at 37°C. Samples were transferred in triplicate to 96-well plates and scanned on the Tecan GENios Rainbow absorbance plate reader (Tecan, San Jose, CA). Data are shown as the mean ± standard deviation (SD) and represent 3 samples per experiment for 4 experiments measured in triplicate. 66  Chapter 2 H9c2-Neo or ARC stable cell lines, L5 and L24, were treated with 0-500 JIM H202 for 8 h. Cell viability was assessed using the calcein-acetoxymethyl (AM) cell viability assay which utilized non-fluorecent dye that is converted to a green-fluorescent calcein after AM ester hydrolysis by intracellular esterases. Calcein is retained in cells with intact membranes (Molecular Probes, Eugene, OR). Briefly, calcein-AM was added to cells at a final concentration of 5 pi.M. Fluorescence intensity was read following one hour (h) of substrate incubation (excitation 485 nm, emission 527 nm) using the'PECAN GENios fluorescent plate reader (Tecan, San Jose, CA).  23.7 H9c2 stable and transient transfection Stably transfected H9c2 cells with pcDNA3-Neo or pcDNA3-ARC were obtained from Dr. Daryoush Ekhterae. 12 Cells were prepared using lipofectamine, as previously described, resulting in the production of Neo-transfected cells and two clones, L5 and L24, which express high levels of ARC. 12 An adenoviral vector expressing ARC (Ad-ARC) and an adenoviral vector expressing green fluorescent protein (Ad-GFP), control, were kindly provided by Dr. Roberta Gottlieb. Adenoviral infections were carried out by removal of media and addition of a 10:1 virus to cell ratio in low volume media. Cells were incubated with intermittent gentle rocking for 2 h, media levels were restored and cells were incubated overnight to allow for protein expression.  67  Chapter 2  23.8 H9c2 myocyte differentiation Muscle differentiation was induced by culturing cells to 100% confluency, removing media containing serum, washing cells twice with DPBS and replacing medium with DMEM containing 1% horse serum (HS). Media was changed daily for 5 days. Differentiation was assessed by measuring expression of differentiation markers by Western blot and was quantified by determining the number of cells that showed at least three nuclei. Multinucleation is expressed as a percentage of the total number of nuclei in ten randomly chosen microscopic fields.  2.3.9 F-actin and nuclear staining of H9c2 cells H9c2 cells transfected with ARC or vector alone were grown on glass coverslips and differentiated as described in section 2.3.8. Cells were stained with AlexaFluor 488-labelled phalloidin and Hoechst 33342 (Molecular Probes, OR) to visualize F-actin and nuclei, respectively. Phalloidin is a water soluble bicyclic peptide derived from Amanita phalloides mushrooms that binds strongly to F-actin. 13 Hoechst is a cell soluble, blue-fluorescent bisbenzimidazole derivative that binds to the minor groove of DNA. 14 Cells were washed twice with DPBS, fixed with 2% paraformaldehyde for 10 min at room temperature and rewashed twice with DPBS. Cells were then permeabilized in a 0.1% Triton X solution for 5 min and washed twice with DPBS. Cells were incubated with 1 pM AlexaFluor 488-labelled phalloidin and 1 pg/m1 Hoechst 33342 for 30 min, washed three times with DPBS before imaging. Slides were imaged using a Nikon Eclipse T6300 microscope and Spot digital 68  Chapter 2 camera. The excitation and emission wavelengths for AlexaFluor 488 and Hoechst 33342 are 350 nm and 461 nm, respectively.  2.3.10 DEVDase activity assay  DEVDase activity assays were performed to detect caspase-3/7-like activity, as described previously. 15 ' 16 At day 0, 1, 3 and 5 post-differentiation, H9c2 cells transfected with ARC or vector alone were lysed in whole cell lysis buffer (1% NP-40, 20 mM Tris, pH 8, 137 mM NaCI, 10% glycerol, 1 mM phenylmethyl sulfoxide, 0.15 U/ml aprotinin, and 1 mM sodium orthovanadate). Lysates (0.3 mg/ml) or buffer as control were plated in triplicate and incubated at 37°C for 15 min. Acetyl-DEVD-7-amino-4-methylcoumarin (Ac-DEVDAMC) (37.5 mg/ml, Calbiochem) caspase-3 substrate was added and relative florescence units (RFU) were measured after 2 h at 37°C using the 1 ECAN GENios fluorescence plate reader (ex: 380 nm, em: 460 nm).  2.3.11 Statistical analysis  All results are expressed as mean ± SE, and analyzed with GraphPad Prism 4 software using one-way analysis of variance (ANOVA) with multiple comparisons performed by Students' T test. The results of statistical tests were considered statistically significant at p<0.05.  69  Chapter 2  2.4 Results 2.4.1 Native ARC expression in endothelial and smooth muscle cell lines  Native levels of ARC expression were assessed in cultured HUVECs and HCASMCs by RT-PCR and Western blot. We were able to detect transcript for ARC in both cell lines. By comparing RNA levels to those of 18S rRNA we observed that ARC transcript levels are higher in HUVECs than in HCASMCs. A representative image of ARC transcript levels is shown in Figure 2.2(A). We then compared protein expression levels of ARC in these cell lines. Again we were able to detect ARC expression in both lines with higher expression levels observed in HUVECs. A representative Western blot is shown in Figure 2.2(B).  2.4.2 TAT-ARC purification and transduction in vascular cells  ARC is a splice variant of the Nop30 protein. Although they have poor homology at the protein level, as a result of a frame shift cause by an alternate splicing at exon 2, there is only one unique sequence of 10 nucleotides contained in ARC that is not in the sequence of Nop30. This sequence is unfortunately a poor target for siRNA. Thus we decided to examine the effect of increased ARC levels via TAT-fusion protein transduction. TAT-ARC andTAT0-gal were expressed in BL21(DE3)pLysS bacteria, purified using Ni-NTA columns and were then desalted. We routinely obtained purified protein concentrations greater that 2 mg/ml. Texas-red conjugated TAT-ARC and TAT-0-gal were successfully transduced into both HCASMCs and HUVECs in culture (Figure 2.3). The transduced protein appears  70  Chapter 2  A  RT-PCR ARC 18S  SMC EC  B  Western blot ARC SMC^EC  Figure 2.2 HCASMCs and HUVECs express ARC. (A) Representative RT-PCR experiment (of n=3) showing detection of ARC transcripts in cultured HCASMCs (SMC) and HUVECs (EC). (B) Representative Western blot (of n=3) demonstrating ARC protein expression.  71  Chapter 2  HCASMC  HUVEC  TAT-ARC  Untreated  Figure 2.3 TAT-ARC fusion protein transduction into HCASMCs and HUVECs. Representative fluorescent images of HCASMCs and HUVECs were treated with Texas-red conjugated TAT-ARC fusion protein demonstrating successful protein uptake.  72  Chapter 2 punctate (Figure 2.4(A)) and was taken up in a concentration-dependent manner (Figure 2.4(B)).  2.4.3 ARC over-expression does not protect against H202 treatment. ARC' s ability to protect against H 20 2 treatment was measured in both HUVECs and HCASMCs. Cells were pre-treated with TAT-ARC or TAT-13-gal (control) and were then exposed to 0.6 mM H202 for 4 h. Viability was measured using the MTS assay (see Figures 2.5 and 2.6). H202 treatment induced an average viability loss in HUVECs of 95.6 ± 1.8% compared to untreated cells and an average viability loss in HCASMCs of 90.8 ± 8.5% compared to untreated cells. In both cell lines TAT-ARC transduction was protective against H202 treatment; however, high levels of transduction (>100nM) were required to see any protective effect and TAT-ARC treatment was no more protective than treatment with the TAT-13-gal fusion protein.  2.4.4 Functional overexpression of ARC in pre-differentiated H9c2 cells Since ARC has previously been shown to be protective in cardiomyocytes and skeletal muscle4 ' 5' 12.17 we turned to the rat embryonic myocyte cell line H9c2. H9c2 cell lines stably overexpressing ARC or Neo (control) were created by selecting clones from pcDNA3 transfected cells. Two clones, L5 and L24, were selected due to their high expression levels of ARC (Figure 2.7 (A)). Both clones were used in subsequent analyses in order to reduce the possibility that the effects observed are resulting from a gene disruption  73  Chapter 2  B  TAT-ARC 411.1111.1111110  ^  f3-actin  0^62.5^125^250^500^1000  TAT-ARC treatment (nM) Figure 2.4 TAT-ARC uptake into HUVECs and HCASMCs is punctate and concentration-dependent. (A) High magnification fluorescent image of Texas-red conjugated TAT-ARC internalization into HUVECs showing punctuate protein distribution. (B) Western blot of ARC protein following TAT-ARC transduction into HUVECs showing concentration-dependent uptake against [3-actin protein control.  74  Chapter 2  120  100  0 80 0  0 "6 60  0 TAT-ARC  :acrs^40  TAT-13-gal  20  0^50^100^250^500 Concentration of TAT fusion protein (nM) -  Figure 2.5 TAT-ARC does not confer greater protection against H20 2 in HUVECs than TAT-13-gal control HUVECs were pretreated with increasing concentrations of TAT-ARC and TAT-13-gal and subjected to 0.6 mM H202 for 4 h. Data are shown as percent viability of untreated cells as measured by the MTS viability assay. Data represents the mean ± SD of 4 experiments, 3 repeats/experiment.  75  Chapter 2  140 120 0 C 0  100  U 0  80  0 TAT-ARC  60  E TAT-0-gal  40 20 0 0^50^100^250  ^  500  Concentration of TAT-fusion protein (nM)  Figure 2.6 TAT-ARC does not confer greater protection against H202 in HCASMCs  than TAT-13-gal control  HCASMCs were pretreated with increasing concentrations of TAT-ARC and TAT-13-gal and subjected to 0.6 mM H202 for 4 h. Data are shown as percent viability of untreated cells as measured by the MTS viability assay. Data represents the mean ± SD of 4 experiments, 3 repeats/experiment. Significance was calculated using a Student's t-test, p-values > 0.1.  76  Chapter 2 as a consequence of vector integration into the host cell genome rather than from the overexpression of ARC. The functionality of ARC was confirmed in these cells lines by examining cell viability following exposure to hydrogen peroxide (Figure 2.7 (B)). H9c2Neo control cells demonstrated concentration-dependent loss in viability after 8 h of treatment with 11202 concentrations between 0 and 500 RM. The H9c2-ARC clones, L5 and L24, demonstrated a significant anti-apoptotic effect at all 11202 concentrations (p-values < 0.05) and were able to maintain cell viability; showing a slight decrease in viability at only the highest concentration of 500 i.iM (73.9 ± 4.2% for L5, 82.5 ± 12.5% for L24, compared to 41.1 ± 4.0% of Neo cells; expressed as mean ± SE).  2.4.5 Characterization of H9c2-ARC cell differentiation We then wanted to examine the role of ARC overexpression in differentiated H9c2 cells. We induced myotube differentiation using standard protocols by reducing serum concentration from 10% FCS to 1% HS. Cells were visualized at 0, 3 and 5 days after the induction of differentiation. H9c2-Neo cells demonstrated myoblast elongation/differentiation; however, elongation was attenuated in H9c2-ARC cells (Figure 2.8). Elevated myotube disarray and disorganization were observed at day 3 and 5 in the ARC overexpressing cells compared to that of the H9c2-Neo cells. In addition to myotube elongation, multi-nucleation was also observed in the H9c2-Neo at a rate of 11.2 ± 5.1%, but was absent in the H9c2-ARC cells (Figure 2.9 and 2.10). To further assess the influence of ARC overexpression on myoblast differentiation, the status of muscle specific proteins myogenin and troponin T were evaluated. Myogenin and troponin T were both highly expressed in differentiated (Day 3 and 5 post-differentiation) 77  Chapter 2  A  H9c2 Neo^L5^L24 - ARC  1111 IN  - 13-actin  B  ^ ■  140 _ 120 _  H9c2/Neo H9c2/L5 H9c2/L24  100 _ 80 60 40 20 0 0^31.3^62.5^125.0^250.0^500.0  H202 concentration (11M)  Figure 2.7 Overexpression of ARC prevents H202-induced cell death. (A) ARC stable cell lines, L5 and L24, express high levels of ARC. (B) H9c2-Neo and ARC cells were treated with various concentrations of H202 and cell viability was assessed using the calcein-AM cell viability assay at 8 h post-treatment. Values are expressed as the percent viability the test group compared to that of untreated H9c2-Neo cells. Bars represent the mean ± SE (n = 3).  78  Chapter 2  Day 0  ^  Day 3  ^  Day 5  Figure 2.8 Overexpression of ARC prevents myogenic differentiation. Representative morphological changes of Neo control and ARC overexpressing stable cell lines, L5 and L24, at day 0, 3 and 5 following the induction of differentiation. Characteristic alignment and elongation of cells is observed in control cells but is not apparent in ARC overexpressing cell lines.  79  Chapter 2  Figure 2.9 Overexpression of ARC prevents myogenic differentiation. Fluorescent staining of ARC overexpressing H9c2 cells at day 5 post-differentiation. H9c2 cells transfected with ARC, or vector alone were stained with AlexaFluor 488-labelled phalloidin for F-actin (green). Cell nuclei were counterstained with Hoechst (blue). Arrows indicate multi-nucleated cells.  80  Chapter 2  18 16 14 H 12 0 Ca  "2 1  10 8 6 4 2 0  Neo  ^  ARC  Figure 2.10 Overexpression of ARC prevents myogenic differentiation. Myogenic differentiation was quantified by the number of differentiated cells which showed at least three nuclei and expressed as a percentage of the total number of nuclei in ten randomly chosen microscopic fields. The results shown are mean ± SD (n=10) and significance was determined by Student's t-test. (*=p<0.05).  81  Chapter 2 H9c2-Neo cells but were minimally detectable in the ARC-overexpressing, L5 and L24, H9c2 cell lines (Figure 2.11). Consistent with these findings, when H9c2-Neo cells were transiently transduced with an adenovirus ARC construct, the muscle differentiation markers were significantly reduced (Figure 2.12).  2.4.6 ARC expression during differentiation We then examined the native expression levels of ARC throughout H9c2 cell differentiation. ARC was undetectable in undifferentiated H9c2 cells. However, ARC expression rose to detectable levels by day 3 following differentiation and by day 5 ARC levels had reached maximal and sustained expression levels (Figure 2.13).  2.4.7 Caspase-3 activation during H9c2 differentiation To examine whether caspase-3 is activated during myotube differentiation, we measured caspase-3 activity by the cleavage of Ac-DEVD-AMC substrate. As shown in Figure 2.14, at day 0 post-differentiation, caspase-3 activity was not different between Neo and ARC overexpressing cells (Neo: 4400 ± 504 RFU, ARC: 4192 ± 2672 RFU; expressed as mean ± SD). However at day 1 post-differentiation caspase-3 activity significantly increased in the Neo cells, implying an important role of caspase activation in cell differentiation. Overexpression of ARC prevented caspase-3 activation at day 1 postdifferentiation (16108 ± 1135 RFU for ARC cells compared to 39736 ± 1796 RFU for Neo cells). By day 3 post-differentiation caspase-3 activity levels decreased in both groups (Neo: 10505 ± 694 RFU, ARC: 14861 ± 1928 RFU) and were relatively stable through to day 5 (Neo: 10303 ± 624 RFU, ARC: 10390 ± 1707 RFU). 82  Chapter 2  Day 0 Neo L5 L24  Day 3  Day 5  Neo L5 L24  Neo L5 L24 -Troponin T  -Myogenin  -ARC -13-actin  Figure 2.11 ARC stable overexpression prevents the expression of the muscle-specific markers troponin T and myogenin. At day 0, 3 and 5 post-differentiation, cell lysates were collected and Western blotting was performed to determine troponin T, myogenin and ARC expression. The same membranes were blotted with an antibody against 13-actin to illustrate equal protein loading. The data are representative of three different experiments.  83  Chapter 2  Day 0^Day 3^Day 5 Ad- Ad- Ad- Ad- Ad- AdGFP ARC GFP ARC GFP ARC -Troponin T  -Myogenin -ARC -I3-actin  Figure 2.12 Transient ARC overexpression prevents the expression of the musclespecific markers troponin T and myogenin. At day 0, 3 and 5 post-differentiation, cell lysates were collected and Western blotting was performed to determine troponin T, myogenin and ARC expression. The same membranes were blotted with an antibody against 13-actin to illustrate equal protein loading. The data are representative of three different experiments.  84  Chapter 2  Neo  0^3^5^7 Days post-differentiation -ARC  swam airmii 4iggie Ma.  -I3-actin  Figure 2.13 ARC levels increase in H9c2 cells upon differentiation. Western blot of ARC expression at day 0, 3 and 5 following the induction of differentiation. The same blot was stained with an antibody against f3 -actin to illustrate equal protein loading.  85  Chapter 2  P<0.005  M 4500 LL  CC  4000 3500  •  3000  ^  II ARC  (,) 2500  CCI •  CI  LIU  Neo  2000 1500 10 00 -  500 0  =it Day 0^Day 1^Day 3^Day 5  Figure 2.14 ARC overexpression prevents caspase-3/7 activity during differentiation. At day 0, 1, 3, and 5 post-differentiation, cell lysates were harvested and caspase activity was determined. The data shown are mean ± SD (n=3) and significance was determined by Student's ttest.  86  Chapter 2  2.5 Discussion ARC expression was initially thought to be restricted to the highly differentiated tissues of skeletal and cardiac muscle. 1 Since that time ARC has also been found to be expressed in other cells types, most notably cancer cells.  18,19  Here we demonstrate that ARC  is also expressed in endothelial cells and smooth muscle cells. Although ARC expression is usually associated with muscle phenotypes we observed greater levels of both ARC transcript and protein expression in endothelial cells compared to smooth muscle. Interestingly, protein transduction of ARC into these cells types did not confer greater protection than the control protein 13-gal to treatment with H202. It is unclear why TAT-ARC is not exerting a protective effect in these cells. One possibility is that TAT-ARC is not being properly folded or transported and thus is not biologically active in these cells. TAT-ARC has been shown to be protective against oxidative injury in cardiac tissue s and ARC overexpression has been shown to protect against H202 treatment in other cell types 12 and as shown here in H9c2 cells. We have also found that although ARC is expressed in differentiated skeletal and cardiac muscle that its expression must be repressed in these tissues to allow differentiation to occur. Apoptosis is a critical physiological process that is essential for normal tissue development and homeostasis. Dysregulation of this form of cell death is associated with numerous pathological conditions. Recent studies suggest that apoptosis and differentiation share common pathways in muscle cells. 2°-22 Actin fibre disassembly and reorganization are conserved features of both apoptosis and myoblast differentiation. Similarly, caspase activity is a key component of both apoptosis and 87  Chapter 2 skeletal muscle differentiation. 2° Caspase-3 inhibition reduces myotube/myofibre formation as well as expression of muscle-specific proteins during myogenesis. 2° Further, dysregulated myoblast differentiation and apoptosis in response to certain cytokines may be associated with increased muscle wasting in chronic disease states such as infection, Acquired Immune Deficiency Syndrome (AIDS) and cancer. 23 Interestingly, tumour necrosis factor alpha (TNFa), a principle cytokine associated with cachexia, is involved in the regulation of skeletal muscle differentiation and apoptosis. 23 Thus, it is exciting to speculate that proteins which inhibit both differentiation and apoptosis could attenuate such degenerative diseases by preventing muscle differentiation thereby facilitating further replication. As such, the role of ARC in the regulation of myocyte differentiation is interesting. H9c2 myoblasts, isolated from rat embryonic cardiomyocytes, proliferate under normal conditions and are mono-nucleated. This cell line is also a well-characterized model of differentiation. When H9c2 cells are exposed to reduced serum concentrations at confluence, they fuse and differentiate into elongated, multi-nucleated myotubes. 24 Although this cell line exhibits some differences from primary cells, H9c2 cells share many of the properties of primary cardiomyoblasts and skeletal muscle 24-27 and can be easily propagated and stably transfected, making them an ideal model for this study. ARC is highly expressed in terminally differentiated cardiac and skeletal muscle cells. 1 However, we were unable to detect ARC expression in undifferentiated H9c2 rat myoblast cells leading us to ask questions regarding the regulation of ARC expression during myoblast differentiation and the importance of this regulation on the differentiation process.  88  Chapter 2 ARC expression increased by day 3 following differentiation and reached maximal and sustained levels by day 5. To understand whether regulation of ARC expression during differentiation is important to the process of differentiation itself, we examined the effect of ARC over-expression using pre-differentiated H9c2 cells. H9c2 cells overexpressing ARC were unable to differentiate as indicated by morphological characteristics such as myotube elongation and multinucleation. Myogenic differentiation, characterized by cell growth arrest, myoblast alignment, elongation, and fusion of mono-nucleated myoblasts into multi-nucleated myotubes, is dependent on the expression of the MyoD family of basic helix-loop-helix (bHLH) transcription factors, which includes MyoD, MyfS, myogenin, and MRF4.  28  Upon  stimulation, myoblasts are induced to express muscle regulatory factors, which in turn leads to the expression of muscle-specific genes. To further assess the influence of ARC overexpression on myoblast differentiation, the status of muscle specific proteins myogenin and troponin T were evaluated. Myogenin and troponin T were both highly expressed in differentiated (day 3 and 5 post-differentiation) H9c2-Neo cells but were minimally detectable in the ARC-overexpressing, L5 and L24, H9c2 cell lines (Figure 2.11). These results were confirmed using H9c2-Neo cells that were transiently transduced with an adenovirus ARC construct. Thus, inhibition of ARC expression is vital for the proper differentiation of these cells. Whether premature ARC expression culminates in abnormal cardiovascular development is unclear and requires further investigation. Recent studies have suggested that caspase-3 activity is required for skeletal muscle differentiation. 20 As ARC inhibits caspase-mediated apoptosis, we examined whether caspase-3 is activated during myotube differentiation and whether overexpression of ARC 89  Chapter 2 prevents that activation. Caspase-3 activity significantly increased at day 1 postdifferentiation and ARC overexpression prevented caspase activation. As ARC does not interact directly with caspase-3, it is likely modifying caspase-3 activation through its interactions with caspase-2, 8 or the Bcl-2 protein Bax, as has previously been shown. 3 ' 29 These findings suggest that ARC expression must be attenuated or absent to allow for early differentiation events including caspase-3 activation and that it is at this early stage of differentiation during which ARC impacts the differentiation process. Our initial finding that ARC is endogenously expressed at undetectable levels until day 3 post-differentiation (i.e. after caspase-3 is activated) supports this hypothesis. Therefore, the inhibition of differentiation markers, such as myogenin and troponin-T, in the ARC overexpressing cell lines are likely a result of these upstream events. This hypothesis would explain why endogenous expression of ARC at day 3 and day 5 does not prevent differentiation. The induction of ARC following caspase-3 activation would likely be beneficial as it may protect properly differentiating cells from apoptosis. It is becoming increasingly more apparent that ARC is a multi-faceted anti-apoptotic protein. Several mechanisms have been proposed as to how ARC attenuates apoptosis including: inhibition of caspases, mitochondrial membrane depolarization, DISC formation, Bax activation, and cytochrome c release or K+ currents.  1-5 ' 12  The results of this study  demonstrate a novel role for ARC in myoblast differentiation and suggest that ARC expression is tightly controlled throughout the differentiation process in order to allow for initiating events such as caspase-3 activity. The challenge for future studies will be to further delineate the detailed mechanisms by which ARCs expression is regulated and contributes to myocyte differentiation as well as the 90  Chapter 2 potential role of ARC in myocyte disarray and disease. Reduced troponin T expression, which we have shown can be induced by ARC expression during differentiation, contributes to myocyte disarray 3° and mutations in this gene are associated with myocyte disarray and 15% of all cases of familial hypertrophic cardiomyopathy. 31 Myofibrillar disarray is also commonly associated with familial hypertrophic cardiomyopathy, the leading cause of sudden death in young athletes, indicating a potential role for ARC in its pathogenesis. The studies shown here indicating ARC is unable to protect vascular cells against oxidant injury induced by H20 2 are preliminary in nature. It is possible that ARC is able to protect against oxidant stress induced through other pathways. Since we were able to detect ARC expression in both endothelial and smooth muscle cell lines it seems likely that ARC does play a role in these cells. One possibility is that the native levels of ARC are high enough to confer maximal protection via its many pathways. Although this is possible, other cell types, such as cardiomyocytes, that express much higher levels of ARC do still gain an additional protective effect upon TAT-ARC transduction. 5 Our ultimate research goal is to examine the relationship between ischemic injury and CAV. Given the intense interest by other groups in examining the role of ARC in myocardial ischemic injury and our failure to obtain preliminary evidence demonstrating a protective role for ARC in vascular ischemic injury we selected to examine other proteins. One such group of proteins are the cytochrome p450 2C enzymes. The role of these enzymes in peri-transplant ischemic injury and CAV are examined in the subsequent chapters of this thesis.  91  Chapter 2  2.6 Bibliography Koseki T, Inohara N, Chen S, et al. ARC, an inhibitor of apoptosis expressed in skeletal muscle and heart that interacts selectively with caspases. Proc Natl Acad Sci USA. Apr 28 1998;95(9):5156-5160. 2. Neuss M, Monticone R, Lundberg MS, et al. The apoptotic regulatory protein ARC (apoptosis repressor with caspase recruitment domain) prevents oxidant stressmediated cell death by preserving mitochondrial function. J Biol Chem. Sep 7 2001;276(36):33915-33922. 3. Nam YJ, Mani K, Ashton AW, et al. Inhibition of both the extrinsic and intrinsic death pathways through nonhomotypic death-fold interactions. Mol Cell. Sep 24 2004;15(6):901-912. 4. Ekhterae D, Lin Z, Lundberg MS, et al. ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis in heart-derived H9c2 cells. Circ Res. Dec 9 1999;85(12):e70-77. 5. Gustafsson AB, Sayen MR, Williams SD, et al. TAT protein transduction into isolated perfused hearts: TAT-apoptosis repressor with caspase recruitment domain is cardioprotective. Circulation. Aug 6 2002;106(6):735-739. 6. Schwarze SR, Ho A, Vocero-Akbani A, et al. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science. Sep 3 1999;285(5433):15691572. 7. Becker-Hapak M, McAllister SS, Dowdy SF. TAT-mediated protein transduction into mammalian cells. Methods. Jul 2001;24(3):247-256. 8. Wadia JS, Dowdy SF. Protein transduction technology. Curr Opin Biotechnol. Feb 2002;13(1):52-56. 9. Wadia JS, Dowdy SF. Modulation of cellular function by TAT mediated transduction of full length proteins. Curr Protein Pept Sci. Apr 2003;4(2):97-104. 10. Nagahara H, Vocero-Akbani AM, Snyder EL, et al. Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kipl induces cell migration. Nat Med. Dec 1998;4(12):1449-1452. 11. Janknecht R, de Martynoff G, Lou J, et al. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Nall Acad Sci USA. Oct 15 1991;88(20):8972-8976. 12. Ekhterae D, Platoshyn 0, Zhang S, et al. Apoptosis repressor with caspase domain inhibits cardiomyocyte apoptosis by reducing K+ currents. Am J Physiol Cell Physiol. Jun 2003 ;284(6):C1405 -1410. 13. Wieland T. Interaction of phallotoxins with actin. Adv Enzyme Regul. 1976;15:285300. 14. Portugal J, Waring MJ. Assignment of DNA binding sites for 4',6-diamidine-2phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study. Biochim Biophys Acta. Feb 28 1988;949(2):158-168. 15.^Granville DJ, Cassidy BA, Ruehlmann DO, et al. Mitochondrial release of apoptosisinducing factor and cytochrome c during smooth muscle cell apoptosis. Am J Pathol. Jul 2001;159(1):305-311. 1.  92  Chapter 2 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.  Granville DJ, Shaw JR, Leong S, et al. Release of cytochrome c, Bax migration, Bid cleavage, and activation of caspases 2, 3, 6, 7, 8, and 9 during endothelial cell apoptosis. Am J Pathol. Oct 1999;155(4):1021-1025. Gustafsson AB, Gottlieb RA, Granville DJ. TAT-mediated protein transduction: delivering biologically active proteins to the heart. Methods Mol Med. 2005;112:8190. Mercier I, Vuolo M, Madan R, et al. ARC, an apoptosis suppressor limited to terminally differentiated cells, is induced in human breast cancer and confers chemoand radiation-resistance. Cell Death Differ. Jun 2005;12(6):682-686. Wang M, Qanungo S, Crow MT, et al. Apoptosis repressor with caspase recruitment domain (ARC) is expressed in cancer cells and localizes to nuclei. FEBS Lett. Apr 25 2005;579(11):2411-2415. Fernando P, Kelly JF, Balazsi K, et al. Caspase 3 activity is required for skeletal muscle differentiation. Proc Natl Acad Sci U S A. Aug 20 2002;99(17):11025-11030. Kageyama K, Ihara Y, Goto S, et al. Overexpression of calreticulin modulates protein kinase B/Akt signaling to promote apoptosis during cardiac differentiation of cardiomyoblast H9c2 cells. J Biol Chem. May 31 2002;277(22):19255-19264. van den Eijnde SM, van den Hoff MJ, Reutelingsperger CP, et al. Transient expression of phosphatidylserine at cell-cell contact areas is required for myotube formation. J Cell Sci. Oct 2001;114(Pt 20):3631-3642. Coletti D, Yang E, Marazzi G, et al. TNFalpha inhibits skeletal myogenesis through a PW1-dependent pathway by recruitment of caspase pathways. Embo J. Feb 15 2002;21(4):631 -642. Kimes BW, Brandt BL. Properties of a clonal muscle cell line from rat heart. Exp Cell Res. Mar 15 1976;98(2):367-381. Hescheler J, Meyer R, Plant S, et al. Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart. Circ Res. Dec 1991;69(6):1476-1486. Kolodziejczyk SM, Walsh GS, Balazsi K, et al. Activation of JNK1 contributes to dystrophic muscle pathogenesis. Curr Biol. Aug 21 2001;11(16):1278-1282. Pagano M, Naviglio S, Spina A, et al. Differentiation of H9c2 cardiomyoblasts: The role of adenylate cyclase system. J Cell Physiol. Mar 2004;198(3):408-416. Buckingham M. Molecular biology of muscle development. Cell. Jul 15 1994;78(1):15-21. Gustafsson AB, Tsai JG, Logue SE, et al. Apoptosis repressor with caspase recruitment domain protects against cell death by interfering with Bax activation. J Biol Chem. May 14 2004;279(20):21233-21238. Peschiaroli A, Figliola R, Coltella L, et al. MyoD induces apoptosis in the absence of RB function through a p21(WAF1)-dependent re-localization of cyclin/cdk complexes to the nucleus. Oncogene. Nov 21 2002;21(53):8114-8127. Sehnert AJ, Huq A, Weinstein BM, et al. Cardiac troponin T is essential in sarcomere assembly and cardiac contractility. Nat Genet. May 2002;31(1):106-110.  93  Chapter 3: Cytochrome p450 2C Contributes to Post-Ischemic Vascular Dysfunction 2  3.1 Introduction Cardiac I/R contribute to MI and is also a major complication in surgical treatments such as cardiac transplantation, balloon angioplasty and coronary bypass. I/R is associated with a burst of ROS which is thought to contribute to both vascular dysfunction and myocardial damage. CYP enzymes, reviewed in section 1.4, are membrane-bound heme-containing terminal oxidases responsible for the oxidation, peroxidation and/or reduction of a large number of substances including cardiovascular drugs. Although the majority of CYP are found in hepatic tissues, these enzymes are also expressed in extra-hepatic tissues such as the intestine, kidney, lung, heart, and blood vessels. CYP enzymes contribute to the cellular generation of ROS. 1 Superoxide anions (02'), hydrogen peroxide and hydroxyl radicals are produced during the CYP reaction cycle when electrons for the reduction of the central heme iron are transferred to the activated bound oxygen molecule.' Recent evidence indicates an important role for CYP in the pathogenesis of several cardiovascular diseases. 2-8 We recently reported that CYPs are key generators of superoxide during I/R in the heart. 3 Furthermore, we demonstrated that CYP 2C9 inhibitors significantly reduce infarct size in both a rat Langendorff perfusion model of global ischemia as well as in a rabbit coronary ligation 94 2  A version of this chapter has been published. Hunter AL, Bai N, Laher I, Granville DJ. (2005). Cytochrome p450 2C inhibition reduces post-ischemic vascular dysfunction. Vascul. Pharmacol. 43: 213-219.  Chapter 3 model of focal ischemia. In the latter study, CYP inhibition also reduced post-ischemic CK release and superoxide generation while restoring coronary flow.  3  In a clinical setting,  sulfaphenazole (SP) restored endothelium-dependent vasodilator responses in patients with manifest coronary artery disease. 2 However, the role of CYP 2C9 in I/R-mediated vascular dysfunction has not been examined. SP, a highly specific inhibitor of CYP 2C9 in humans, exerts its inhibitory effect by binding to the heme group of CYP 2C9. Its specificity is mediated through interactions between SP's phenyl substituent and the phenyl group of Phe 114 on CYP 2C9. 9 SP also inhibits CYP 2C6 in rats. 10 SP is dose dependently specific for CYP 2C9 (humans) and CYP 2C6 (rats).  3.2 Aim As CYP 2C6/9 inhibition increases endothelium-dependent vasodilation in patients with coronary artery disease and increases post-ischemic coronary flow we hypothesized that CYP 2C6/9 inhibition will decrease endothelium-dependent vasodilation following I/R. The aim of this study was to assess the role of the CYP 2C6/9 inhibitor SP on endotheliumdependent, NO-mediated vasodilation in post-ischemic vascular dysfunction. We demonstrate for the first time that SP restores endothelium-dependent vascular function following I/R.  95  Chapter 3  3.3 Materials and Methods 3.3.1 Heart perfusion and vessel cannulation  Experimental protocols were approved by the Animal Care Committee of the University of British Columbia. A copy of the animal care certificate is provided in Appendix I. Male Sprague-Dawley rats (300-350 g, n=10) were injected with sodium pentobarbital (60 mg/kg) and heparin sulphate (1000 U/kg) intraperitoneally. After loss of reflexes in rats, hearts were removed and immediately placed in ice-cold modified Krebs' buffer (composition in mM: NaCI 119, KC1 4.7, KH2PO4 1.18, NaHCO3 24, MgSO47H2O 1.17, CaC12 1.6, and glucose 11.1). Hearts were perfused in the Langendorff mode as previously described. 3 Hearts were randomly divided into three groups: i) I/R: hearts were perfused 20 min followed by 30 min no-flow global ischemia and 15 min reperfusion with modified Krebs' buffer, ii) I/R with SP (I/R+SP): hearts prepared as in the li/R group but with the addition of SP (10 p.M) in the perfusate, and iii) control: hearts were perfused for the total perfusion time of I/R groups without ischemic period (35 min) with modified Krebs' buffer. After perfusion (control) or reperfusion (I/R, I/R+SP), hearts were removed from the Langendorff apparatus and placed in a dissection dish with ice-cold buffer. Septal coronary arteries (intraluminal diameter is between 190-290 tun at 20 mm Hg) were dissected and transferred to the chamber of a pressure myograph. A Video Dimension Analyzer (Living Systems Instrumentation, Burlington, VT, USA) was used to measure inner diameter as described elsewhere.11  96  Chapter 3 3.3.2 Vasomotor responses in septal arteries  Septal coronary arteries were pretreated with U46619 (1 tiM, Cayman Chemical, Ann Arbor, MI) at 20 mm Hg. After a sustained constriction, tissues were exposed to ACh (1 nM10 uM) added to the external reservoir, and final maintained diameters were recorded. An identical protocol was used to study the vasodilator effects of sodium nitroprusside (SNP, 1 nM-10 uM), and isoproterenol (1 nM-10 uM). In addition, the constrictor responses to various concentrations of KC1 (20, 35, 66, 84 mM) were examined. At the end of each experiment, Krebs' buffer was substituted with Krebs' buffer containing no CaC12 and 2.0 mM EGTA to achieve zero calcium and the maximal passive diameters.  3.3.3 Dihydroethidium (DHE) staining of coronary blood vessels  Hearts were flash frozen in liquid nitrogen following Langendorff perfusion, as described above, and stained for superoxide production modified from methods previously described (Miller et al., 1998). Frozen hearts were sectioned at 20 ptm on a ThermoShandon cryostat. DHE (Molecular Probes, Eugene, OR) was prepared under  N2 gas  by dissolving to 1  mg/m1 in DMSO and then dilution in phosphate buffered saline (PBS). Sections were treated in 2 tiM DHE for 30 min at 37°C under N2 gas. Sections were washed twice with PBS, cover slips were applied and slides and were imaged immediately. Imaging was performed on an Eclipse TE300 fluorescent microscope (Nikon; excitation: 488 nm, emission: 610 nm) under identical exposure settings. Fluorescence density of arterial walls (n=6, control; n=5, SP) were quantified using Image-Pro Plus software. Values were normalized to the average of the arteries from the untreated hearts representing 100%. 97  Chapter 3 3.3.4 Measurements of dityrosine in coronary effluents  Dityrosine measurements were performed utilizing the method developed by Yasmin et  a/. 12 L-tyrosine (0.3 mM) was added to Kreb's buffer and Langendorff perfusions were  carried out as described above. Coronary effluent fractions were collected. Upon reaction with peroxynitrite, L-tyrosine is converted to dityrosine which absorbs at 320 nm.  3.3.5 Statistical analysis  All results are expressed as mean ± SE, and analyzed with NCSS 2000 and PASS 2000 software using one-way analysis of variance (ANOVA) and/or repeated-measures ANOVA with multiple comparisons performed by Bonferroni's test. -LogEC50 (pD2) was calculated by Graphpad Prism®, version 3.02. The results of statistical tests were considered statistically significant at p<0.05.  3.4 Results 3.4.1 Endothelium-dependent vasomotor responses  Tonic contractions for U46619 (1 JAM), a stable analog of thromboxane  A2,  were not  altered in either I/R or 1/R+SP groups as compared to those obtained under control conditions. Reduced endothelium-dependent vasodilation was observed after 1/R. Figure 3.1 shows  98  Chapter 3 290  A  Control  174  a) 250  E 4 -  I/R+ 10 [tM SP  141 227  .44) 1-1  134  I/R U46619 Wash  5 min  B  O Control O I/R  •  I/R+10 pM SP  Log [ACh] M Figure 3.1. Sulfaphenazole (SP) restores post-ischemic endothelium-dependent NOmediated vasodilation. (A) Representative traces showing SP attenuates impaired endothelium-dependent vasorelaxation to ACh. (B) Concentration-response curves to ACh, showing that SP increased sensitivity and maximal responses to ACh (n=5, *p<0.05). 99  Chapter 3 representative traces (A) and response curves (B) demonstrating a rightward shift in the acetylcholine (ACh) concentration-response curve following I/R. The pD2 for ACh was 7.2 ± 0.1 (control) and 6.6±0.1 (I/R, n=5, p<0.005). Sensitivity to ACh was restored by SP (10 pM) with a pD 2 of 7.3±0.1 (n=5, p<0.005, I/R+SP vs. I/R).  3.4.2 Endothelium-independent vasomotor responses Vasodilator responses to SNP were also reduced after I/R (Figure 3.2A, B). SP was not able to reverse this impairment. The maximal responses elicited by SNP in control, I/R, and I/R+SP were 85.7 ± 5.1%, 68.9 ± 3.2%, 67.9 ± 8.3%, respectively (n=4, p<0.01, control vs. UR and I/R+SP). Likewise, SP (10 JIM) failed to restore I/R-induced impairment of isoproterenol-mediated vasodilation (Figure 3.3A, B). The maximal responses elicited by isoproterenol in control, I/R, and I/R+SP were 93.2 ± 1.3%, 76.3 ± 2.6%, 75.5 ± 3.0%, respectively (n=4, p<0.01, control vs. I/R and I/R+SP). Vasoconstriction elicited by KCl demonstrated no significant differences in control (Figure 3.4), I/R and I/R+SP groups (maximal responses, which were produced at 66 mM KC1, were 57.8 ± 2.3%, 57.3 ± 5.4%, and 58.6 ± 5.7%, respectively).  3.4.3 Post-ischemic ROS production DHE staining measures ROS production and is primarily reactive with superoxide. DHE staining was performed to assess superoxide production in arterial walls following I/R (Figure 3.5 and 3.6). Pre-treatment with 10 tiM of SP caused a significant reduction in the  100  Chapter 3  A  2811 $.• 4.4 H ,.. W 233 cts "t:J 243 73  Control  •g 161  I/R+ 1011M SP  1  UR  151 _ U46619 ^ ^ Wash SNP (1 nM — 10µM) 5 min  B^100 80  C  60  O  x 40  w  cc  s) 20  r •^ cl Control 0 I/R • I/R+10 uM SP  "■•  .  -7 Log[SNP] M  Figure 3.2. SP does not restore post-ischemic endothelium-independent vasodilation produced by sodium nitroprusside (SNP). (A)Traces showing impaired vasodilation to SNP in UR and UR+SP groups. (B) Concentration-response curves to SNP (n=4) showing that SP does not improve endotheliumindependent vasodilation. 101  Chapter 3  A  217  Control  I 1120  g 90  I/R+10 tiM SP  V 505 30  UR  1-1169 U4661 9  5 min  ^ Isoproterenol Wash (1 nM — 10 ptM)  B 100 80 60 40 20  ^ Control O I/R  •  I/R+1 0 pM SP  -9^-8^-7^-6 Log [Isopoterenol] M Figure 3.3 SP does not restore post-ischemic endothelium-independent vasodilation produced by isoproterenol. (A)Traces showing impaired vasodilation to isoproterenol in UR and UR + SP groups. (B) Concentration-response curves to isoproterenol (n=5), showing that SP does not improve endothelium-independent vasodilation. 102  Chapter 3 80 O 60 :a7 0 • 40 O  C.) o4! 20 0  -20  --c"— Control — 0-- I/R 1113+10 pM SP -  0  20^40^60 [KCI] M  ^ ^ 80 100  Figure 3.4 Constrictor responses to KC1 were unaffected by SP pre-treatment. Response curves to KC1 indicating that vasoconstriction was unaffected by I/R and SP had no added effect (n=5, p>0.95).  103  Chapter 3  PBS Control  ^  Dihydroethidium  Untreated  10 pM SP  -  Figure 3.5 SP reduces ROS production following I/R. Representative traces demonstrating that SP reduces relative fluorescent intensity of dihydroethidium staining following I/R.  104  Chapter 3  120 100 80  *  60  I^  40 20 0  Untreated  ^  10µM SP  Figure 3.6 SP reduces ROS production following UR. Mean ± SE of the fluorescent intensity of dihydroethidium staining following UR in arterial walls (control n=6, SP n=5, * p<0.005).  105  Chapter 3  E 0.04 0.03 C)  0.02 ii v  0.01  10  5  15  Time (min)  Figure 3.7 Peroxynitrite measurements in post-ischemic coronary effluent. Dityrosine conversion from L-tyrosine as measured by absorbance at 320 nm. L-tyrosine was added to the perfusate during Langendorff-perfusion induced I/R.  106  Chapter 3 relative intensity of DHE staining to 40.2 ± 6.4% of untreated hearts 100.0 ± 5.9% (n=5, p<0.005, vs. control n=6).  3.5 Discussion Increased intracellular calcium levels following I/R induces the activation of phospholipase A2 (PLA2) and the subsequent hydrolysis of AA from membrane phospholipids. 13-16 AA is metabolized by CYP 2C9 into EETs, however, superoxide is also generated during this reaction cycle.'' Superoxide readily reacts with NO• to produce ONOO- and it has been proposed that NO , scavenging due to CYP 2C9-mediated superoxide production leads a reduction of NO• bioavailability that impairs endothelium-dependent vascular function in atherosclerosis. 2 Inhibition of iPLA2 or PLA2 protects against I/R, further supporting the link between AA and superoxide generation. 15  18 19 '  '  Thus, given that  SP restores endothelium-dependent, NO-mediated vasodilation in patients with coronary artery disease 2 in combination with our recent findings that SP significantly reduces infarct size caused by I/R 3 , we hypothesized that SP attenuates post-ischemic endothelial dysfunction. Although the rat equivalent of CYP 2C9 has not been fully characterized, a CYP 2C9-like isozyme that shares immunoreactivity and is selectively inhibited by SP has been detected in rat arteries. 3 ' 20 CYP 2C6 is a putative rat homologue for human CYP 2C9. In support of this, SP has been shown to inhibit rat CYP 2C6 but not other members of the rat CYP 2C family. 1°  107  Chapter 3 Here we showed that SP reduced the FR-induced loss of endothelium-dependent, NO•-mediated vasodilation to ACh. However, SP was not able to improve the marked postischemic impairment of endothelium-independent vasodilation (SNP, isoproterenol). Fichtlsherer et al. 2 also observed a similar trend where SP had no effect on impaired responses to SNP in patients with coronary artery disease versus normal controls. Impairment of endothelium-independent vasodilation was not due to a loss in smooth muscle cell contractility as responses to KCl were similar in all three treatment groups. These results indicate that SP is acting through an endothelium-specific mechanism. SP's inability to restore responses to either SNP or isoproterenol, indicate that it is not acting by altering guanylate or adenylate cyclase activities, but more likely acting to restore endothelium NO• bioavailability. Our data suggests that inhibition of CYP 2C6/9 increases vasodilation through a reduction in superoxide formation and consequent increase in NO• bioavailability. Figure 3.8 shows a diagram of the proposed mechanism. Chloramphenicol, a potent of inhibitor of CYP 2C6/9, has previously been shown to reduce superoxide production in the heart following I/R. However, it was unclear what effect CYP 2C6/9 inhibition had on post-ischemic vessel wall ROS production. To examine this question, superoxide formation was assessed by staining with DHE following FR with or without SP assessed. DHE is converted to ethidium in the presence of ROS and is most highly reactive with superoxide. Consequent ethidium staining is visible by fluorescent microscopy. DHE conversion was quantified in the area around vascular walls. There was a significant reduction (-60%) of superoxide in vessels of hearts pretreated with SP. These results indicate that CYP 2C9 contributes to FR-induced vascular  108  Chapter 3  I/R Injury 0,01111111181.101011040111110M141111$011110 0001111111$1111111101111011101 111101001011W11111101110 11140110$1110114114114trif  i[Ca2i]c ---.Phospholipase A 2  Arachidonic acid  ON00EET and 20-HETE  11$1:14111141111011111*111001110011101111144111:01$11101111111:14:01:14011411:101:14$111e1.401011111111111021*:41)1011101101$11111$111011101  1111.111,41.1101111, ItItititiglitititilltItatitit10141104111;111111111111111011$101111111$111111#11111111011110111101411141$111;1411:1101n$14:4101110113$01141$11111$101111  SMC Vasodilation Figure 3.8 Proposed mechanism of CYP 2C induced impaired post-ischemic vasodilation. Upon I/R, [Ca 2 1, increases activating phospholipase A2 hydrolysis of arachidonic acid from phospholipid membranes. AA is metabolized by CYP 2C leading to the production of EETs and 20-HETE and 02 . 02 then reacts with NO• forming ONOO- and reducing the bioavailability of NO• and its ability to induce vasodilation. ONOO- can also inhibit vasodilation. -.  109  Chapter 3 superoxide production and supports the hypothesis that CYP 2C9 mediates post-ischemic vascular dysfunction by reducing NO• bioavailability. SP has previously been shown to have no effect on complex I, II, and IV of mitochondrial respiration and does not reduce superoxide generated via NADPH oxidase or xanthine oxidase. 1 ' 3 Therefore, SP is not acting as a general antioxidant and is likely acting through specific inhibition of CYP 2C6/9; a known producer of superoxide. Inhibition of ROS production following UR has been examined using several experimental models that employ superoxide dismutase and antioxidants. Although these studies have demonstrated the role of ROS in I/R they have not resulted in the development of effective treatments to alleviate I/R injury. SP presents a promising alternative strategy at reducing ischemic injury as we have shown that it significantly decreases superoxide production and improves vascular function. Treatment with SP is particularly promising in the context of cardiac surgical procedures such as cardiac transplantation, balloon angioplasty and coronary bypass where I/R is predictable and SP could be administered prior to the ischemic period. In summary, we report novel findings indicating that that i) I/R impairs both endothelium-dependent and independent vasodilation, ii) SP selectively restores postischemic endothelium-dependent, NO-mediated vasodilation and iii) SP reduces UR-induced superoxide production. Our study indicates that SP confers a protective effect in postischemic vascular dysfunction through a reduction of CYP 2C6/9-mediated superoxide production. Thus, CYP 2C9 is a potentially important therapeutic target for patients with ischemic heart disease and those undergoing surgical procedure where UR-injury is a factor.  110  Chapter 3  3.6 Bibliography 1. 2. 3. 4. 5. 6. 7. 8. 9.  10. 11.  12. 13. 14.  Fleming I, Michaelis UR, Bredenkotter D, et al. Endothelium-derived hyperpolarizing factor synthase (Cytochrome P450 2C9) is a functionally significant source of reactive oxygen species in coronary arteries. Circ Res. Jan 19 2001;88(1):44-51. Fichtlscherer S, Dimmeler S, Breuer S, et al. Inhibition of cytochrome P450 2C9 improves endothelium-dependent, nitric oxide-mediated vasodilatation in patients with coronary artery disease. Circulation. Jan 20 2004 ;109(2): 178-183. Granville DJ, Tashakkor B, Takeuchi C, et al. Reduction of ischemia and reperfusioninduced myocardial damage by cytochrome P450 inhibitors. Proc Natl Acad Sci U S A. Feb 3 2004;101(5):1321-1326. Gross ER, Nithipatikom K, Hsu AK, et al. Cytochrome P450 omega-hydroxylase inhibition reduces infarct size during reperfusion via the sarcolemmal KATP channel. J Mol Cell Cardiol. Dec 2004 ;37(6): 1245-1249. Hunter AL, Cruz RP, Cheyne BM, et al. Cytochrome p450 enzymes and cardiovascular disease. Can J Physiol Pharmacol. Dec 2004;82(12):1053-1060. Wang XL, Greco M, Sim AS, et al. Effect of CYP1A1 MspI polymorphism on cigarette smoking related coronary artery disease and diabetes. Atherosclerosis. Jun 2002; 162(2):391-397. Wu S, Chen W, Murphy E, et al. Molecular cloning, expression, and functional significance of a cytochrome P450 highly expressed in rat heart myocytes. J Biol Chem. May 9 1997;272(19):12551-12559. Yasar U, Bennet AM, Eliasson E, et al. Allelic variants of cytochromes P450 2C modify the risk for acute myocardial infarction. Pharmacogenetics. Dec 2003; 13(12):715-720. Melet A, Assrir N, Jean P, et al. Substrate selectivity of human cytochrome P450 2C9: importance of residues 476, 365, and 114 in recognition of diclofenac and sulfaphenazole and in mechanism-based inactivation by tienilic acid. Arch Biochem Biophys. Jan 1 2003;409(1):80-91. Kobayashi K, Urashima K, Shimada N, et al. Selectivities of human cytochrome P450 inhibitors toward rat P450 isoforms: study with cDNA-expressed systems of the rat. Drug Metab Dispos. Jul 2003;31(7):833-836. Skarsgard PL, Wang X, McDonald P, et al. Profound inhibition of myogenic tone in rat cardiac allografts is due to eNOS- and iNOS-based nitric oxide and an intrinsic defect in vascular smooth muscle contraction. Circulation. Mar 21 2000;101(11):1303-1310. Yasmin W, Strynadka KD, Schulz R. Generation of peroxynitrite contributes to ischemia-reperfusion injury in isolated rat hearts. Cardiovasc Res. Feb 1997;33(2):422-432. Freyss-Beguin M, Millanvoye-van Brussel E, Duval D. Effect of oxygen deprivation on metabolism of arachidonic acid by cultures of rat heart cells. Am J Physiol. Aug 1989;257(2 Pt 2):H444-451. Leong LL, Sturm MJ, Ismail Y, et al. Plasma phospholipase A2 activity in clinical acute myocardial infarction. Clin Exp Pharmacol Physiol. Feb 1992 ;19(2): 113-118.  111  Chapter 3 15. 16. 17.  18. 19. 20.  Van der Vusse GJ, Reneman RS, van Bilsen M. Accumulation of arachidonic acid in ischemic/reperfused cardiac tissue: possible causes and consequences. Prostaglandins Leukot Essent Fatty Acids. Jul 1997;57(1):85-93. Williams SD, Gottlieb RA. Inhibition of mitochondrial calcium-independent phospholipase A2 (iPLA2) attenuates mitochondrial phospholipid loss and is cardioprotective. Biochem J. Feb 15 2002;362(Pt 1):23-32. Fulton D, McGiff JC, Wolin MS, et al. Evidence against a cytochrome P450-derived reactive oxygen species as the mediator of the nitric oxide-independent vasodilator effect of bradykinin in the perfused heart of the rat. J Phannacol Exp Ther. Feb 1997;280(2):702-709. Ogata K, Jin MB, Taniguchi M, et al. Attenuation of ischemia and reperfusion injury of canine livers by inhibition of type II phospholipase A2 with LY329722. Transplantation. Apr 27 2001;71(8):1040-1046. Sargent CA, Vesterqvist 0, McCullough JR, et al. Effect of the phospholipase A2 inhibitors quinacrine and 7,7-dimethyleicosadienoic acid in isolated globally ischemic rat hearts. J Pharmacol Exp Ther. Sep 1992;262(3):1161-1167. Earley S, Pastuszyn A, Walker BR. Cytochrome p-450 epoxygenase products contribute to attenuated vasoconstriction after chronic hypoxia. Am J Physiol Heart Circ Physiol. Jul 2003;285(1):H127-136.  112  Chapter 4: Cytochrome p450 2C Contributes to Cardiac Allograft Vasculopathy 3 4.1 Introduction More than 3,000 heart transplants are performed worldwide annually. Current immunosuppressive regimens are very effective in preventing acute rejection. Unfortunately, chronic rejection associated with CAV remains a major hurdle to long-term graft survival of all vascularized organ transplants. CAV is an accelerated and diffuse form of arteriosclerosis that can be detected in up to 75% of heart transplant recipients following the first year of transplantation." Although immunological mechanisms clearly play an important role in the pathogenesis of CAV, non-immunological mechanisms, such as peri-transplant UR injury, also contribute via direct damage or indirectly through cross-talk with immune responses associated with this type of vasculopathy. 3 ' 4 The transplant organ is vulnerable to UR injury induced by graft ischemia time, quality of graft preservation during transport, hemodynamic status of the donor, catecholamines used for inotropic support, and reperfusion itself. 5 Compelling evidence supports a molecular and cellular basis for a causal relationship between UR injury during transplantation and the onset and progression of CAV. 6 ' 7 UR injury to endothelial cells may provide the initial trigger for atherogenesis by stimulating platelet adhesion, release of growth factors, upregulation of MHC Class I and II expression, release of donor antigens, expression of adhesion molecules, and proliferation of vascular smooth muscle cells. (Reviewed 113 3  A version of this chapter has been submitted for publication. Hunter AL, Kerjner A, Mueller KJ, McManus BM and Granville DJ. (2008). Cytochrome p450 2C enzymes contribute peri-transplant ischemic injury and cardiac allograft vasculopathy. Am J Transplant.  Chapter 4 1n3-91  Thus, attenuation of I/R injury would be of great benefit to transplant recipients not only  through the inhibition of direct cellular injury, but also indirectly through the aforementioned factors that influence the allo-immune response. Several experimental models using superoxide dismutase and antioxidants have demonstrated the importance of ROS in the pathophysiology of I/R injury 1 " 5 ; however, the development of effective treatments to alleviate reperfusion injury remains elusive. CYPs, as described in section 1.4, are membrane-bound heme-containing terminal oxidases that exist in a multi-enzyme system that includes a FAD/FMN-containing NADPH cytochrome p450 reductase and cytochrome b5. The CYP superfamily is responsible for the oxidation, peroxidation and/or reduction of vitamins, steroids, cholesterol, xenobiotics and the majority of cardiovascular drugs in an oxygen and NADPH-dependent manner. Although the vast majority of CYP are found in hepatic tissues, other CYP have been shown in recent years to be expressed predominantly in extra-hepatic tissues such as the heart, blood vessels, gut, kidney and lung. The role of CYP in cardiovascular disease is poorly understood, increasing evidence suggests that these enzymes play a role in the pathogenesis of a number of cardiovascular diseases. 16-24 Previously, we discovered that the rat equivalent of CYP 2C9 makes a significant contribution to superoxide generation and cell death associated with I/R • 21 injury. Recently, we demonstrated that the CYP 2C inhibitor sulfaphenazole (SP) increases  endothelium dependent vasodilation and decreases vascular superoxide production following ischemia and reperfusion. 25  114  Chapter 4  4.2 Aim CYP 2C enzymes contribute to post-ischemic vascular dysfunction and cell death. As ischemic injury is thought to contribute to CAV, we hypothesized that CYP 2C will contribute to the development of CAV. The aim of this study was to investigate whether CYP 2C inhibition during the peri-transplant period would reduce post-transplant oxidative damage and vascular remodelling associated with chronic cardiac rejection.  4.3 Materials and Methods 43.1 Heterotopic heart transplantation  All protocols were designed in accordance with the guidelines, and approved by, the animal care committee of the University of British Columbia. A copy of the animal care certificate for heterotopic heart transplantation is provided in Appendix I. Minor histocompatibility antigen-mismatched rat heterotopic heart transplants were performed between male Lewis donor (RT1 1 , 260-330 g) and Fisher 344 recipient (RT1 1 ", 230-280 g) rats. All rats were purchased from Charles River Laboratories (Wilmington, MA) and cardiac transplantation was performed as previously described. 1 Donors and recipients were treated with 5 mg/kg SP intraperitoneally (IP, Clinalpha, Laufelfingen, Switzerland), or vehicle control 1 hr prior to surgery. Donors were anaesthetized with xylazine (10 mg/kg)/ketamine (120 mg/kg), IP. The inferior vena cavae (WC) were isolated, slowly perfused with heparinized saline and clamped distally. The right and left superior vena cavae (SVC) were then ligated. The ascending aortas were cut below the brachiocephalic artery and the main 115  Chapter 4 pulmonary arteries were cut proximal to their bifurcations. They were flushed with heparinized saline. Pulmonary veins were ligated together and the donor hearts were gently detached and placed in ice-cold heparinized saline. Recipients were anaesthetized with isofluorane (4% induction, 2% maintenance) Anastomoses were performed between the ascending aortas of donor hearts and the abdominal aortas and between the pulmonary arteries and the inferior vena cavae of the recipient animals. Buprenophrine was administered subcutaneously at 0.01 mg/kg immediately following surgery. A copy of the standard operating procedure outlining the detailed protocol for this operation is provided in Appendix II.  4.3.2 Tissue collection  At 4, 7 and 30 days post-transplantation the animals received heparin (50 U/kg, IP) and were anaesthetized with a combination of ketamine hydrochloride (120 mg/kg) and xylazine hydrochloride (10 mg/kg). Thoracic and abdominal cavities were opened and transplanted hearts were assessed for heartbeat. The circulatory system was flushed by injecting 25 ml of Ringer's buffer at 80 mmHg into the right ventricle and cutting a small incision in the right atria to allow fluids to drain. Rats were then perfusion fixed by replacing Ringer's with 4% formalin 80 mmHg and allowing it to circulate as above. The native and transplanted hearts were then removed rapidly, and transverse sections immersion fixed in 10% formalin for 24 h before being embedded in paraffin.  116  Chapter 4 4.3.3 Histological staining and immunohistochemistry (IHC)  Formalin-fixed, paraffin-embedded sections were stained with hematoxylin and eosin (H&E) and Movat's pentachrome stain using standard methods. IHC was performed on formalin-fixed, paraffin embedded ventricular transverse sections. Briefly, sections were deparaffinized by baking in a 60°C oven for 1 h followed by serial rehydration by immersion in 100% xylene (3X 5 min), 100% ethanol (2X 5 min), 90% ethanol (3 min), 70% ethanol (3 min) and Tris-buffered saline (TBS, pH 7.4, 2X 5 min). Antigen retrieval was performed by boiling sections in citrate-citric acid buffer (pH 6.0) for 15 min, allowing sections to reach room temperature and washing 2X with TBS. Exogenous phosphates were quenched by incubation in 10% H202 in TBS for 10 min and washing 2x with TBS. Sections were blocked by incubation of sections with blocking buffer (10% normal serum of the species the secondary antibody was raised in) for 30 min. Sections were incubated in primary antibodies overnight in blocking buffer at 4°C. Primary antibodies utilized were: 1:50 monoclonal a-rat Ki-67 clone M1B-5 and 1:100 polyclonal a-human CD3 (Dako Canada, Missisagua, ON), 1:100 monoclonal a-rat CD8 MRC OX-8 (Genetex, San Antonio, TX), and 1:800 polyclonal a-Von Willebrand Factor (Abcam Inc, Cambridge, MA). Sections were then washed 3X in TBS + 0.01% Tween 20 (TBST). Secondary detection was performed for 1 hr at room temperature with 1:350 biotinylated anti-secondary antibodies (Vector Laboratories, Burlingame, CA) in blocking buffer supplemented with 3% normal rat serum. The addition of 3% normal rat serum was required to reduce cross reactivity with rat IgG. Staining was visualized using the ABC kit (Vector Laboratories) followed by detection with the chromagens Nova Red or diaminobenzidine tetrahydrochloride (DAB, Vector Laboratories,  117  Chapter 4 Burlingame, CA), and nuclei were counterstained with hematoxylin. Slides were coverslipped using Aqua-mount ® aqueous mountant (Lerner Laboratories, Pittburgh, PA).  4.3.4 Histological assessment and quantification  Four micrometer sections were stained with H&E or Movat's pentachrome stain. H&E-stained sections of 30 day post-transplant sections were scored on a 0-5 scale (five rats per group, n=7 arteries/rat) for general, focal, sub-epicardial, sub-endocardial, and perivascular immune infiltration. Luminal narrowing in all visible medium to large size coronary arteries (30 days post-transplantation, n = 5 rats per group, 3 sections per rat, 7 arteries per section) was evaluated on Movat's pentachrome stained sections. Briefly, ImagePro PlusTM  (MediaCybernetics, Silver Spring, MD) was used to quantify intimal and luminal  areas, and percent luminal narrowing was calculated as the area of the lumen as a percentage of the combined area of the lumen and the intima. For assessments of immune infiltration the vascular wall area was defined as the region from the lumen to the outside of the medial smooth muscle layer and the perivascular space (PVS) was defined as the region between the medial smooth muscle layer and the myocardium. These regions were traced and quantified using Image-Pro P1 us TM.  4.3.5 Luminex analysis  Blood was collected in accordance with the guidelines, and approved by, the animal care committee of the University of British Columbia. A copy of the animal care certificate 118  Chapter 4 for blood collection is provided in Appendix I. Tail vein blood was collected into heparinized tubes 1 day prior to transplant and at days 1, 3, 5 and 7 post-transplant. Serum was isolated by allowing blood to clot for 30 min and centrifuging for 15 min at 1000 g. Samples were analyzed using the rat cytokine/chemokine premixed LINCOplex 14-plex premix bead kit as per manufacturer's recommendations (LINCO Research, St. Charles, MO). Briefly, serum samples were diluted 1:5 in LINCO Serum MatrixTM and standards were diluted in four-times serial dilutions to 1:4096. The assay filter plate was blocked using LINCO Assay buffer for 10 min at room temperature and was fluid was removed by gentle vacuum filtration. Diluted samples, standards and controls were incubated with premixed cytokine/chemokine detection beads overnight with agitation at 4°C at which time samples were drained by gentle vacuum filtration and washed two times with LINCO washing buffer. Plates were developed by the addition of the detection antibody cocktail and the streptavidin-phycoerythrin detection solution. Upon gentle vacuum filtration, plates were washed two times with wash buffer and bead-antibody complexes were solubilized in sheath fluid for analysis. Plates were analyzed on the Luminex FlowMetrix System (Qiagen, Mississauga, ON). Sample parameters required a minimum of 50 events per bead. Samples were run in duplicate and compared to an 8-point standard curve developed using a 5-parameter logistic fit graph.  4.3.6 8-Isoprostane measurements Free 8-isoprostane measurements were performed using the 8-isoprostane EIA Kit on left ventricular blood samples collected at sacrifice on day 4 and 7 post transplantation as per manufacturer's recommendations (Cayman Chemical Company, Ann Arbor, MI). Briefly, 119  Chapter 4 samples were collected into tubes containing EDTA and 0.005% BHT (butylated hydroxytoluene) was added to prevent further production of 8-isoprostanes in the samples. Samples were stored at -80°C until time of analysis. Samples were then purified using 8isoprostane affinity sorbent purification kit (Cayman). An equal volume of 15% KOH was added and samples were incubated at 40°C for 60 min. Four volumes of ethanol containing 0.01% BHT was added and samples were vortexed, incubated for 5 min on ice and centrifuged for 10 min at 1500 g. Supernatants were collected and ethanol was evaporated under nitrogen to less than 10% vol/vol. Purification required neutralizing the samples to pH 7.4 by addition of 2 volumes of 1 M KH2PO4 and 1 volume of eicosanoid affinity column buffer. Samples were then added to pre-equilibrated 8-isoprostane affinity sorbent and allowed to bind for 60 min at room temperature, centrifuged briefly at 1500 g and supernatant was discarded. Sorbent was then washed twice with ultrapure water and 8isoprostanes were retrieved by incubating sorbent in ethanol Elution Solution with vortexing. Samples were dehydrated using a speed vacuum system and resuspended in EIA buffer for ELISA. Purified samples and 8-isoprostane standards were then incubated with EIA Buffer, AChE tracer and 8-isoprostane antiserum on the EIA plate for 18 h at room temperature. EIA plates were developed by the addition of Ellman's reagent and 8-isoprostane tracer followed by incubation for 90 min at room temperature. Samples were measured via absorbance readings at 412 nm on the Tecan GENios Rainbow absorbance plate reader (Tecan, San Jose, CA).  120  Chapter 4 4.3.7 Statistical analysis Repeated measures general linear model analysis was performed for Luminex results using SPSS Statistical Software (Chicago, IL). Wilks' Lambda multivariate tests were utilized to assess significance. For other assays, statistical differences between two groups were determined using a Student's t-test. For both tests, a p-value (alpha error) of 0.05 or less was considered significant.  4.4 Results The heterotopic heart transplant model was utilized to assess the contribution of CYP 2C to peri-transplant I/R injury and the development CAV. Donor hearts were transplanted from Lewis donor rats into the abdominal cavities of Fisher 344 recipients by suturing the ascending aorta of donor hearts and the recipients abdominal aorta and the donor's pulmonary artery to the recipients inferior vena cava. This represents a minor histocompatability mismatch allowing us to assess vascular changes associated with chronic rejection.  4.4.1 Post-surgical morbidity and mortality Donor and recipient rats were treated with 5 mg/kg of SP or saline control 1 h prior to surgery in order to assess the contribution of CYP 2C to cardiac rejection. SP is a specific inhibitor of CYP 2C9 in humans and is a potent inhibitor of CYP 2C6, a putative homologue 121  Chapter 4 of human CYP 2C9, in rats. 26 Surgical and post-surgical morbidity and mortality rates were similar and very low (i.e. <5%) in both SP and control groups. Post-surgical recovery was also similar in both groups with recipients regaining their pre-surgical weigh by 9.2 ± 2.9 days in the SP group versus 10.3 ± 4.0 days in the control group (Figure 4.1). Transplant recipients were euthanized and organs were harvested at days 4, 7 and 30 post-transplant. At harvesting, all transplanted hearts had palpable heart beats.  4.4.2 Assessment of CYP 2C6 expression in rat heart cross-sections Initially, immunohistochemical studies were performed to confirm expression of CYP 2C6 in the transplanted hearts. Results demonstrate positive staining for CYP 2C6 in both the myocardium and vasculature of transplanted hearts (Figure 4.2).  4.4.3 CYP 2C contributes to luminal narrowing in rat heterotopic heart transplants Transplanted hearts harvested at day 30 were then utilized to assess the development of CAV by measuring the degree of luminal narrowing in the large coronary blood vessels (Figure 4.3). Pre-treatment with SP at the time of transplantation resulted in a dramatic decrease in luminal narrowing by day 30 (12.1 ± 4.1% vs. 66.2 ± 13.6% for control, p=0.0002; Figure 4.4).  4.4.4 Assessment of general immune infiltration Thirty day post-transplant hearts were then assessed for characteristics of general immune rejection. H&E-stained sectioned were scored for diffuse, focal, sub-epicardial, subendocardial infiltration (Figures 4.5 and 4.6). SP pre-treatment did not result in a statistically 122  Chapter 4  130'  ± Control -11-SP (5 mg/kg)  80  0^5^10^15^20^25^30 Days post-transplant  Figure 4.1 SP treatment does not reduce post-transplant weight gain. Rats receiving heterotopic heart transplants were weighed prior to transplantation and daily for 30-days post-transplant. Both SP and control groups had characteristic weight loss immediately following surgery followed by gradual weight gain. Values expressed as mean ± SD (n=5). There was no difference between treatment groups (p>0.1).  123  Chapter 4  Myocardium  Blood vessels C 0 0 a) 0)  a)  z  Figure 4.2 CYP 2C6 is expressed in transplanted rat heart blood vessels and myocardium. Paraffin-embedded transplanted hearts from Lewis rats sacrificed 30 days post-surgery were immunohistochemically stained for the presence of CYP 2C6 using Vector NovaRED as a substrate. Primary antibody-absent negative staining controls and CYP 2C6 antibodypositive staining result are shown for transplanted hearts. Scale bar equals 100 vim.  124  Chapter 4  Untreated  ^  SP (5 mg/kg)  = 100 [tm  Figure 4.3 SP administration at time of surgery attenuates allograft luminal narrowing. Representative coronary blood vessels in Movat's pentachrome stained coronary cross sections from rat heterotopic heart transplants 30 days post-transplantation.  125  Chapter 4  100 80-  c 0^  co  60  40 -  z 0  ^20-  No treatment 5 mg/kg SP  Figure 4.4 SP administration at time of surgery attenuates allograft luminal narrowing. Percent luminal narrowing in rat heterotopic heart transplants were performed between Lewis donor and Fisher recipient rats 30 days post-transplantation. Luminal narrowing was measured using Image-Pro Plus as the percent of the area of the lumen divided by the area of the internal elastic lamina. Data are expressed as mean ± SD (n= 5 hearts, 3 sections/heart, 7 vessels/section, * p<0.005).  126  Chapter 4  C  HI .1 00 pm Figure 4.5 Histological features of diffuse and focal myocardial infiltration. H&E stained myocardial sections from 30-day post surgical rat heterotopic heart transplants. Representative images showing (A) normal myocardium, (B) diffuse myocardial infiltration and (C) focal immune myocardial infiltration.  127  Chapter 4  Transplanted heart  Native heart E 1  E )  0 w  E 1  E5 0 0  4rdz - ^1p  'jet kt4.^P f'11) it 4%, 147:40 (  4,  ^k  its  P4P^  -  47  ,1  • •  H  = 100 pm  Figure 4.6 Histological features of epicardial and endocardial immune infiltration. Representative H&E-stained epicardial and endocardial sections from native and 30-day post surgical heterotopic transplanted hearts. Transplanted hearts demonstrate subepicardial and subendocardial immune infiltration.  128  Chapter 4 significant decrease in any of these parameters (Figure 4.7). A trend was observed towards a decrease in both the number of blood vessels with perivascular infiltration and the severity of the infiltration but, again, these findings were not significant (p>0.1; Figure 4.8). Perivascular infiltration was not associated with luminal narrowing with some vessels demonstrating severe perivascular infiltration with minimal luminal narrowing and others demonstrating luminal narrowing with minimal infiltration (Figure 4.9).  4.4.5 CYP 2C does not contribute to T-lymphocyte infiltration T-lymphocyte infiltration was further assessed by IHC staining and quantification in both the vascular wall and the perivascular space (PVS) for general CD3 + T-lymphocytes (Figure 4.10) and for CD8 +-positive cytotoxic T-lymphocytes (Figure 4.11). SP pretreatment did not alter the degree of CD3 + or CD8 + lymphocyte infiltration in either the vascular wall (CD3 + : 3.4 ± 3.6 vs. 2.0 ± 1.2 cells/100 [tm 2 for control, p=0.49; CD8 + : 2.5 ± 1.8 vs. 1.3 ± 1.0 cells/100 j.tm 2 for control, p=0.23) or the PVS (CD3 + : 8.4 ± 3.4 vs. 6.5 ± 2.9 cells/100 p.m 2 for control, p=0.42; CD8 + : 6.6 ± 1.4 vs. 6.4 ± 2.2 cells/100 ii.m 2 for control, p=0.92).  4.4.6 CYP 2C does not significantly alter post-transplant apoptosis Early post-transplant apoptotic events were assessed by measuring TUNEL positivity in the myocardium, endothelium and smooth muscle layers of transplant sections isolated 4 days post-transplantation. TUNEL positivity was lower in SP treated rats in all regions assayed however these differences were not statistically significant (Figure 4.12). Endothelia were stained with Von Willebrand Factor and no loss of endothelial cells was detected (Figure 4.13). 129  Chapter 4  O No treatment ®10 NM SP  Diffuse  ^  Focal^Epicardial^Endocardial  Figure 4.7 CYP 2C does not contribute to general myocardial immune infiltration. Immune infiltration was scored from absent to severe in a blinded fashion in the cardiac muscle. Data are represented as mean ± SD. There was no significant difference between SP and control groups in any immune infiltration category.  130  Chapter 4  5  A 4  3  2  1 0  No treatment  ^  5 mg/kg SP  100  B 80-  60-  40 -  20 -  No treatment^5 mg/kg SP  Figure 4.8 CYP 2C does not contribute to perivascular immune infiltration. Perivascular immune infiltration was assessed in rat heterotopic heart transplants. (A) Immune infiltration was scored from absent to severe in a blinded fashion in the cardiac muscle. (B) The percent of blood vessels with immune infiltration is shown. Percent luminal narrowing expressed as mean ± SD (n= 5 hearts, 7 vessels/heart) p>0.1.  131  Chapter 4  C  Figure 4.9 Perivascular immune infiltration in the absence of luminal narrowing. H&E-stained, paraffin-embedded sections from 30-day post-transplant rats showing coronary blood vessels. (A) Representative image of native blood vessel. (B) Representative image of CAV associated luminal narrowing in control groups. This blood vessel shows significant luminal narrowing with only moderate immune infiltration. (C) Representative image of blood vessel showing severe perivascular immune infiltration in the absence of luminal narrowing. Scale bar equals 100 p.m.  132  Chapter 4  E O O  14 1210  ^ No treatment 0 SP (5 mg/kg)  4CD 5% 8 0  6 4 2  CO  0  Vascular Wall  ^  PVS  Figure 4.10 SP treatment does not alter CD3 + lymphocyte infiltration in the vasculature. Paraffin-embedded sections were immuno-stained for CD3 + lymphocytes. The number of CD3 + cells was quantified using Image-Pro Plus as the number of positive stained cells per area of the vascular wall or the PVS. PVS was defined as the area from the outside of the defined smooth muscle layer to the commencement of the myocardium. Data are represented as the mean ± SD of n=5 rats/group, n=7 vessels/rat.  133  -  Chapter 4  E 10  \i  - 9 o 8 7 ›, 6 _C 5 CL 4  ^ No treatment 0 SP (5 mg/kg)  >, 3 2 co 0 1 0 0 Vascular Wall PVS  Figure 4.11 SP treatment does not alter CDS' lymphocyte infiltration in the vasculature. Paraffin-embedded sections were immuno-stained for CD8 + lymphocytes. The number of CD8 + cells was quantified using Image-Pro Plus as the number of positive stained cells per area of the vascular wall or the PVS. PVS was defined as the area from the outside of the defined smooth muscle layer to the commencement of the myocardium. Data are represented as the mean ± SD of n=5 rats/group, n=7 vessels/rat.  134  Chapter 4  7 —6 w =5c a) > 4w o 3-  O. -J III 2 -  z z 1- 10 :, 0  Myocardium  ^ Endothelium^Smooth muscle  Figure 4.12 CYP 2C does not significantly contribute to post-transplant apoptosis. Paraffin-embedded section from heterotopically transplanted hearts 4 days post-transplant were assessed by TUNEL staining. The percentage of positively stained nuclei relative to total nuclei relative to endothelial, smooth muscle and myocardial cells was measured using Image-Pro Plus. No statistically significant difference between untreated and SP treated transplanted hearts was observed. For endothelial and smooth muscle cell counts were performed on 10 blood vessels/heart, 5 hearts/ group; and for myocardial measurements were performed on 10 fields of view/heart, and 5 hearts/group. Data are expressed as the mean ± SD.  135  Chapter 4  H=100 pm  Figure 4.13 CYP 2C-inhibition has an insignificant effect on EC loss at day 4 posttransplant. Paraffin-embedded section from heterotopically transplanted hearts 4 days post-transplant were assessed by immuno-staining with the endothelial marker von Willebrand Factor and DAB staining. Endothelial loss was not observed in control hearts (A) or those treated with SP (13).  136  Chapter 4  4.4.7 CYP 2C contributes to SMC proliferation following transplantation SMC proliferation was assessed at day 4 following transplantation by counting cells with Ki-67 immunostaining. SP pre-treatment decreased the number of Ki-67 positive SMC (3.3 ± 3.3% for SP vs. 7.2 ± 2.3% for control, p=0.006). Figure 4.14 shows quantification of Ki-67 staining (A) and representative positive staining (B).  4.4.8 Peripheral cytokine and chemokine levels following transplantation Serum which was isolated at 1 day prior to transplantation and at days 1, 3, 5 and 7 post-transplantation was utilized to analyze cytokine levels in the systemic circulation via the Luminex multiplex assays. Serum levels of granulocyte macrophage colony-stimulating factor (GM-CSF), growth-related oncogene (GRO/KC), interferon gamma (IFN-y), monocyte chemotactic protein 1 (MCP-1), and interleukin (Il-) la, 1f3, 2, 10, and 18 were assessed for alterations with time and with time by group. We observed a high level of variability between samples. Quantified cytokine levels are shown in Table 4.1 and a summary table to statistical analyses is shown in Table 4.2. GRO/KC and IL-la,  10, 2, 10  and 18 did not show any significant alterations with time or by group (p>0.1). MCP-1 levels showed a non-significant increase following transplantation (p=0.064) which was similar in both treated and non-treated groups (p=0.755). GM-CSF showed an effect with time (p=0.004), peaking at day 3 post-transplant but there was no significant difference with SP treatment (p=0.239). INF-y levels (Figure 4.15) increased following transplantation in the control group (p=0.023) however this trend was significantly reduced in the SP treated rats (p=0.028). 137  Chapter 4  A 12  C.) 10 C  -  U) 0 0CO  No treatment  ^  SP (5 mg/kg)  B  Figure 4.14 CYP 2C contributes to SMC proliferation. Paraffin-embedded section from heterotopically transplanted hearts 4 days post-transplant were assessed by immuno-staining with the proliferation marker Ki-67 and DAB staining. (A) Quantification was performed by calculating the number of Ki-67 positive nuclei of the total number of nuclei in the defined smooth muscle cell layer. Data are represented as mean ± SD of n=5 rats/group, 7 vessels/rat, * = p<0.05. (B) A representative image of positive Ki67 staining (arrows). Scale bar represent 1001,1m. 138  Chapter 4  cyto-  Days post-transplantation  kine (Willi)  GMCSF GRO/ KC IFN-y MCP1 I1-la 11-113 11-2 I112p70  11-18  -1  CNT  SP  CNT  SP  CNT  SP  CNT  SP  CNT  SP  225± 211 788 ± 103 21 ± 9 218± 101 43± 32 45± 43 747 ± 843 7± 6 211± 153  317± 217 808 ± 59 23 ± 9 183 ± 40 62± 32 31± 13 696 ± 480 18± 10 143± 28  197± 122 1331± 1050 18 ± 21 938 ± 350 52± 6 23± 20 448 ± 230 75± 115 136± 81  114± 58 869 ± 162 4± 7 714 ± 147 13± 15 8± 4 245 ± 113 7.5± 11 98± 51  345± 233 956 ± 386 52 ± 56 870 ± 559 50± 44 49± 44 922 ± 676 23± 17 1486± 1538  162.3± 48.1 601± 131 4± 6 1231 ± 1866 21± 18 9± 2 427 ± 108 423± 139 1017± 1846  230± 109 789± 156 209± 258 600 ± 262 72± 92 26± 25 653 ± 455 267± 319 315± 170  459± 297 675 ± 114 84 ± 46 1294 ± 1961 51± 74 34± 25 1451 ± 973 314± 402 752± 1139  131± 227 79 7± 459 461± 813 445 ± 332 127± 203 21± 24 485 ± 627 1406± 2425 128± 133  79± 93 512 ± 190 1059± 2576 486 ± 377 191± 461 26± 36 312 ± 365 676± 1131 207± 163  Table 4.1 Peripheral cytokine and chemokine levels following heterotopic heart transplantation in SP treated and control rats. Serum was isolated from tail blood vein collected 1 day before transplantation and on days 1, 3, 5 and 7 post-transplant. Samples were assessed for cytokine and chemokine levels using the Luminex assay. Data are shown as mean ± SD in pg/ml for n=5 rats per group.  139  Chapter 4  Wilks' Lambda Multivariate Test (p-value)  Cytokine  Effect with time  Effect of time by group  GM-CSF  0.004*  0.239  GRO/KC  0.068  0.154  IF'N-y  0.023*  0.028*  MCP-1  0.064  0.775  Il- 1 a  0.194  0.557  I1-113  0.602  0.449  11-2  0.315  0.788  11-18  0.301  0.667  Table 4.2 Repeated measures analysis of peripheral cytokine and chemokine levels following heterotopic heart transplantation in SP treated and control rats. Serum was isolated from tail blood vein collected 1 day before transplantation and on days 1, 3, 5 and 7 post-transplant. Samples were assessed for cytokine and chemokine levels using the Luminex assay. Data were analyzed for significant differences utilizing repeated measures multivariate analysis. P-values were calculated using the Wilks' Lambda test. (*=p< 0.05)  140  Chapter 4  700  ■ No treatment  600.^  • SP (5 mg/kg)  --" 500'  65 400.  -  -  sa. *--300' ?..... 1^■ Z 200 u_ 1001^i '  s  ^f a  h  it  .  ii  ^R  .  . ^.  ^!I •  ^2 3 4 5 6 7 8 -2 - 1 - 100-^1  Days Post-transplant  Figure 4.15 CYP 2C contributes to peripheral IFN-y levels post-transplantation. Serum was isolated from tail blood vein collected 1 day before transplantation and on days 1, 3, 5 and 7 post-transplant. Samples were assessed for IFN-y levels (pg/ml) using the Luminex assay. Data were analyzed for significant differences utilizing the repeated measures multivariate analysis. P-values were calculated using the Wilks' Lambda test. IFN-y levels increase with time (p-=0.023) and are significantly reduced in the SP treatment group compared to control (p=0.028).  141  Chapter 4  4.4.9 CYP 2C contributes to serum 8-isoprostane levels Serum isolated at sacrifice (day 4 or 7) was analyzed by EIA to assess oxidative stress via free 8-isoprostane levels. Results, shown in Figure 4.16, demonstrated a peak in 8isoprostane levels at day 4 in the control group compared to the SP treatment group (2746 ± 367 pg/ml for control vs. 1040 ± 181 pg/ml for SP, mean ± SEM, p=0.026). These levels were reduced by day 7 and were similar in both groups (466 ± 62 for control vs. 543 ± 197 pg/ml for SP, mean ± SEM, p=0.389).  4.5 Discussion The findings of this study demonstrate, for the first time, that CYP 2C contributes to peri-transplant ischemic injury. We have found that inhibition of CYP 2C by SP reduces early signs of ischemic injury including oxidative stress and a trend towards a decrease in apoptosis. This finding is consistent with our earlier studies that showed a decrease in vascular and myocardial superoxide production following I/R injury following pre-treatment with SP. 21. 25 CYPs have been previously been shown to significantly contribute to the cellular production of ROS such as 02 . , hydrogen peroxide and hydroxyl radicals during the CYP reaction cycle when electrons for the reduction of the central heme iron are transferred to the activated bound oxygen molecule. 18 We also found a reduction in early (day 4) smooth muscle cell proliferation, as indicated by Ki-67 staining, in rats pre-treated with SP. This corresponds to a significant  142  Chapter 4  3500 3000 g.. 2500 a) o. c 2000 a  ro2  1500 o. o .0 _ 1000 cO 500 0 Day 4  ^  Day 7  Figure 4.16 CYP 2C contributes to post-transplant serum free 8-isoprostane levels. Serum was isolated from left ventricular blood at days 4 and 7 post-transplantation. Serum was analyzed for free 8-isoprostane by ETA. Data represent mean ± SD for n=5 rats/group, samples were run in triplicate.  143  Chapter 4 decrease in intimal thickening and luminal narrowing by day 30 post-transplantation. It was previously hypothesized that SP increases the bioavailability of nitric oxide (NO.) by reducing its reaction with superoxide. In support of this hypothesis, SP is able to increase NO-mediated vasodilation following ischemia/reperfusion injury. 25 Studies by Fleming's group demonstrated that SP enhances endothelium-dependent vasodilator responses in patients with manifest coronary artery disease. The observed effect was suggested to be due to the increased bioavailability of NO- as a consequence of reduced CYP 2C9-mediated superoxide generation and ONOO- formation in endothelial cells. 20 Both reduced NO and elevated ONOO- have important implications in the pathogenesis of CAV and smooth muscle cell proliferation. Endothelial NO- is known to inhibit smooth muscle cell proliferation. 27-3° We also observed an increase in IFN-y levels following transplantation in the control group and that these levels were significantly reduced in the SP treatment group. IFN-y has been shown to induce smooth muscle cell proliferation in a phosphoinositol 3kinase dependent manner. 31 In summary, CYP 2C inhibition during the peri-transplant period prevented development of CAV by inhibiting early SMC proliferation and intimal hyperplasia. This affect could be a result of reduced post-ischemic oxidative damage, contributing to increased NO bioavailability and/or prevention of IFN-y production. As described above, both NO. and IFN-y have previously been shown to contribute to SMC proliferation. Future studies will be geared towards further elucidating the mechanism of CYP 2C-mediated postischemic endothelial dysfunction.  144  Chapter 4  4.6 Bibliography 1. 2.  3.  4. 5. 6.  7.  8. 9. 10.  11.  12.  13.  14.  Dong C, Granville DJ, Tuffnel CE, et al. Bax and apoptosis in acute and chronic rejection of rat cardiac allografts. Lab Invest. Dec 1999 ;79(12) :1643-1653 . Yeung AC, Davis SF, Hauptman PJ, et al. Incidence and progression of transplant coronary artery disease over 1 year: results of a multicenter trial with use of intravascular ultrasound. Multicenter Intravascular Ultrasound Transplant Study Group. J Heart Lung Transplant. Nov-Dec 1995;14(6 Pt 2):S215-220. Wilhelm MJ, Kusaka M, Pratschke J, et al. Chronic rejection: increasing evidence for the importance of allogen-independent factors. Transplant Proc. Aug 1998;30(5):2402-2406. Laskowski I, Pratschke J, Wilhelm MJ, et al. Molecular and cellular events associated with ischemia/reperfusion injury. Ann Transplant. 2000;5(4):29-35. Valantine HA. Cardiac allograft vasculopathy: central role of endothelial injury leading to transplant "atheroma". Transplantation. Sep 27 2003;76(6):891-899. Day JD, Rayburn BK, Gaudin PB, et al. Cardiac allograft vasculopathy: the central pathogenetic role of ischemia-induced endothelial cell injury. J Heart Lung Transplant. Nov-Dec 1995;14(6 Pt 2):S142-149. Schneeberger H, Schleibner S, Inner WD, et al. The impact of free radical-mediated reperfusion injury on acute and chronic rejection events following cadaveric renal transplantation. Clin Transpl. 1993:219-232. Tilney NL, Paz D, Ames J, et al. Ischemia-reperfusion injury. Transplant Proc. FebMar 2001;33(1-2):843-844. Waaga AM, Gasser M, Laskowski I, et al. Mechanisms of chronic rejection. Curr Opin Immunol. Oct 2000;12(5):517-521. Kim J, Kil IS, Seok YM, et al. Orchiectomy attenuates post-ischemic oxidative stress and ischemia/reperfusion injury in mice. A role for manganese superoxide dismutase. J Biol Chem. Jul 21 2006;281(29):20349-20356. Murata S, Miniati DN, Kown MH, et al. Superoxide dismutase mimetic m40401 reduces ischemia-reperfusion injury and graft coronary artery disease in rodent cardiac allografts. Transplantation. Oct 27 2004;78(8):1166-1171. Tanaka M, Mokhtari GK, Terry RD, et al. Overexpression of human copper/zinc superoxide dismutase (SOD1) suppresses ischemia-reperfusion injury and subsequent development of graft coronary artery disease in murine cardiac grafts. Circulation. Sep 14 2004;110(11 Suppl 0:11200-206. Nelson SK, Gao B, Bose S, et al. A novel heparin-binding, human chimeric, superoxide dismutase improves myocardial preservation and protects from ischemiareperfusi on injury. J Heart Lung Transplant. Dec 2002 ; 21(12):1296-1303. Yin M, Wheeler MD, Connor HD, et al. Cu/Zn-superoxide dismutase gene attenuates ischemia-reperfusion injury in the rat kidney. J Am Soc Nephrol. Dec 2001;12(12):2691-2700.  145  Chapter 4 15.  16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.  Abunasra HJ, Smolenski RT, Morrison K, et al. Efficacy of adenoviral gene transfer with manganese superoxide dismutase and endothelial nitric oxide synthase in reducing ischemia and reperfusion injury. Eur J Cardiothorac Surg. Jul 2001;20(1):153-158. Wang X, Zuckerman B, Pearson C, et al. Maternal cigarette smoking, metabolic gene polymorphism, and infant birth weight. Jama. Jan 9 2002;287(2):195-202. Wang XL, Greco M, Sim AS, et al. Effect of CYP1A1 MspI polymorphism on cigarette smoking related coronary artery disease and diabetes. Atherosclerosis. Jun 2002;162(2):391-397. Fleming I, Michaelis UR, Bredenkotter D, et al. Endothelium-derived hyperpolarizing factor synthase (Cytochrome P450 2C9) is a functionally significant source of reactive oxygen species in coronary arteries. Circ Res. Jan 19 2001;88(1):44-51. Fisslthaler B, Popp R, Kiss L, et al. Cytochrome P450 2C is an EDHF synthase in coronary arteries. Nature. Sep 30 1999;401(6752):493-497. Fichtlscherer S, Dimmeler S, Breuer S, et al. Inhibition of cytochrome P450 2C9 improves endothelium-dependent, nitric oxide-mediated vasodilatation in patients with coronary artery disease. Circulation. Jan 20 2004;109(2):178-183. Granville DJ, Tashakkor B, Takeuchi C, et al. Reduction of ischemia and reperfusioninduced myocardial damage by cytochrome P450 inhibitors. Proc Natl Acad Sci U S A. Feb 3 2004;101(5):1321-1326. Yasar U, Bennet AM, Eliasson E, et al. Allelic variants of cytochromes P450 2C modify the risk for acute myocardial infarction. Pharmacogenetics. Dec 2003;13(12):715-720. Wu S, Chen W, Murphy E, et al. Molecular cloning, expression, and functional significance of a cytochrome P450 highly expressed in rat heart myocytes. J Biol Chem. May 9 1997;272(19):12551-12559. Wu S, Moomaw CR, Tomer KB, et al. Molecular cloning and expression of CYP2J2, a human cytochrome P450 arachidonic acid epoxygenase highly expressed in heart. J Biol Chem. Feb 16 1996;271(7):3460-3468. Hunter AL, Bai N, Laher I, et al. Cytochrome p450 2C inhibition reduces postischemic vascular dysfunction. Vascul Pharmacol. Oct 2005;43(4):213-219. Kobayashi K, Urashima K, Shimada N, et al. Selectivities of human cytochrome P450 inhibitors toward rat P450 isoforms: study with cDNA-expressed systems of the rat. Drug Metab Dispos. Jul 2003 ;31(7):833-836. Tanner FC, Meier P, Greutert H, et al. Nitric oxide modulates expression of cell cycle regulatory proteins: a cytostatic strategy for inhibition of human vascular smooth muscle cell proliferation. Circulation. Apr 25 2000;101(16):1982-1989. Ruiz E, Del Rio M, Somoza B, et al. L-Citrulline, the by-product of nitric oxide synthesis, decreases vascular smooth muscle cell proliferation. J Pharmacol Exp Ther. Jul 1999;290(1):310-313. Janssens S, Flaherty D, Nong Z, et al. Human endothelial nitric oxide synthase gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation after balloon injury in rats. Circulation. Apr 7 1998;97(13):1274-1281. Stein CS, Fabry Z, Murphy S, et al. Involvement of nitric oxide in IFN-gammamediated reduction of microvessel smooth muscle cell proliferation. Mol Immunol. Sep 1995;32(13):965-973. 146  Chapter 4 31. Wang Y, Bai Y, Qin L, et al. Interferon-gamma induces human vascular smooth muscle cell proliferation and intimal expansion by phosphatidylinositol 3-kinase dependent mammalian target of rapamycin raptor complex 1 activation. Circ Res. Sep 14 2007;101(6):560-569.  147  Chapter 5: Cytochrome p450 2C9 in Vascular Cell Death and Oxidative Stress 4 5.1 Introduction CYPs are membrane-bound heme-containing terminal oxidases that exist in a multienzyme system that includes a FAD/FMN-containing NADPH cytochrome p450 reductase and cytochrome b5. The CYP superfamily is responsible for the oxidation, peroxidation and/or reduction of vitamins, steroids, cholesterol, xenobiotics and the majority of cardiovascular drugs in an oxygen and NADPH-dependent manner. CYP 2C9 is a monooxygenase that is strongly expressed in the liver and small intestine ) but is also expressed in the heart and the vasculature. CYP 2C9 is an epoxygenase and is involved in the metabolism of arachidonic acid (AA) into epoxyecosotrienoic acids (EETs). Specifically, CYP 2C9 produces 5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET which are involved in NOindependent vasodilation. 2 During ischemia increased intracellular calcium levels induces the activation of phospholipase A2 (PLA2) and the subsequent hydrolysis of AA from membrane phospholipids. 3-6 AA metabolism is further increased during reperfusion. 7 AA is metabolized by three major pathways; the cyclooxygenase (COX) pathway, the lipoxygenase (LOX) pathway, and the CYP epoxygenase pathway, described in detail in section 1.5. AA metabolism during UR has been implicated as a key contributor in the progression of ischemic injury as inhibition of PLA2 or inducible PLA2 (iPLA2) protects against  I/R.5'  8,  9  148 A version of this chapter is in preparation for publication. Arwen L. Hunter, Paul Hiebert, David J. Granville. Cytochrome p450 2C9 expression increases oxidative stress and cell death following hypoxia and reoxygenation in human umbilical venous endothelial cells. 4  Chapter 5 However, several AA metabolites, including cyclooxygenase-2 (COX-2)-derived prostacyclin, are known to be cardioprotective. CYP 2C are the primary epoxygenases involved in AA metabolism by the third pathway.  1°  As previously described, CYP 2C also  make a significant contribution to the cellular production of ROS such as O2', hydrogen peroxide and hydroxyl radicals during AA metabolism. 11 ' 12 Previous studies by our group and others have shown that CYP 2C9, or the rodent or rabbit equivalent, plays a role in vascular function in coronary flow following WR injury 13 vasomotion in patients with coronary artery disease 14 and post-ischemic vascular function' s (see Chapter 3). In recent experiments, described in Chapter 4, we found that CYP 2Cs also contribute to post-transplant oxidative stress, smooth muscle proliferation and CAV development. As AA can be metabolized by one of three possible mechanisms, it is logical that if one of these pathways were blocked, that this may result in a shift towards the other 2 pathways and increased activity of these pathways.  5.2 Aim We hypothesized that CYP 2C9 expression would increase EC death and dysfunction following hypoxia and re-oxygenation (H/R). Further, that CYP 2C9 inhibition would reduce these effects whereas COX inhibition would exacerbate them. The aim of this research is to examine the effect of CYP 2C9 in cultured human endothelial and smooth muscle cells in response to H/R. Specifically, we examined the contribution of CYP 2C9 to cell death in  149  Chapter 5 endothelial and smooth muscle cell lines. We also examined the effect of COX-2 inhibition on CYP 2C9-mediated oxidative stress following H/R.  5.3 Materials and Methods 5.3.1 Cell culture Pooled HUVECs were obtained from Cambrex (Baltimore, MD). HUVECs were cultured in complete endothelial growth medium (EGM: endothelial basal medium supplemented with 0.4% bovine brain extract, 0.1 % human endothelial growth factor (hEGF), 0.1% hydrocortisone and 0.1% gentomycin-amphotericin B (GA-1000); Cambrex) plus 5% foetal bovine serum (FBS, Invitrogen). HCASMCs were cultured in complete smooth muscle growth medium (SmGM: smooth muscle basal medium supplemented with 0.1% insulin, 0.5% human foetal growth factor B, 0.1% GA-1000 and 0.1% hEGF; Cambrex) plus 5% FBS.  5.3.2 Cell lysis and Western blotting for CYP 2C9 Cells were washed twice with ice cold PBS and lysed in CellLytic M lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich, Oakville, ON). Protein concentrations were measured using the Bio-Rad protein assay, which is a modified Bradford protein assay (Bio-Rad, Hercules, CA). This assay measures the change in absorbance of Coomassie Brilliant Blue G-250 to 595nm upon binding to basic and aromatic amino acids in proteins. Equal amounts of protein were separated by sodium dodecyl sulphate — polyacrylamide gel  150  Chapter 5 electrophoresis and then transferred to nitrocellulose membranes. Equal volume amounts of purified human liver microsome (HLM) diluted 1:20 in lysis buffer was used as a control for CYP 2C9 expression. After blocking with 2% skim milk, the membranes were incubated with primary antibodies (1:500 anti-human CYP 2C9 (BD Gentest, San Jose, CA) or 1:1000 monoclonal anti-13-actin (AC-74) antibody (Sigma-Aldrich, St. Louis, MO)) overnight at 4°C, followed by incubation for 1 h with 1:4000 IRDye800 Tm or 1:2000 IRDye700 —conjugated secondary antibodies (Rockland Inc. Gilbertsville, PA). Protein expression was detected by using the Odyssey Infrared Imaging System from LI-COR Biosciences (Lincoln, NE).  5.3.3 Adenoviral expression of CYP 2C9 in HUVEC An adenoviral vector expressing CYP 2C9 sense and an adenoviral vector expressing CYP 2C9 antisense, control, were kindly provided by Dr. Ingrid Fleming. Adenoviral infections were carried out by removal of media and addition of a 10:1 virus to cell ratio in low volume media. Cells were incubated with intermittent gentle rocking for 2 h, media levels were restored and cells were incubated overnight to allow for protein expression.  5.3.4 Optimization of hypoxic conditions Hypoxic conditions were generated in a Coy hypoxic glove box (Coy Laboratories, Grass Lake, MI). This glove box permits control of temperature, humidity, CO2 and 02. Conditions were optimized to generate a P02 between 10 and 20 mmHg, as observed during cardiac ischemia. 16-20 The P02 of the air and of culture media were assessed under 151  Chapter 5 humidified conditions at 37°C and 5% CO2. Media was bubbled with chamber air for 20 s prior to measurements. P0 2 was measured using Oxylab P02 probes and a tissue oxygenation monitor (Oxford optronix, Oxford, UK). 5.3.5 Cell viability in response to H/R  HUVECs transduced with Ad-CYP 2C9 sense or antisense and HCASMCs were seeded in 96-well plates, grown to 90% confluency. Cells were pre-treated with 10 1.1M sulfaphenazole (SP, Clinalpha, Laufelfingen, Switzerland) 1 h prior to induction of H/R. Cells were transferred to the hypoxic chamber, media was removed and cells were washed twice in PBS. Basal media bubbled in low oxygen conditions was then added to cells and cells were exposed to 24 h hypoxia in followed by 4 h of re-oxygenation in normoxic conditions, complete media containing 5% FBS. Viability was assessed using the CellTiter96TM AQueous Assay (MTS) (Promega, Madison, WI). MTS is described in section 2.2.6. MTS was protected from light and was added at a 1:5 ratio of MTS: media and the reaction was allowed to proceed for 1 h at 37°C. Samples were measured in triplicate for absorbance at 490 nm on the Tecan GENios Rainbow absorbance plate reader (Tecan, San Jose, CA). Data are shown as the mean ± SD and represent 3 samples per experiment for 4 experiments measured in triplicate.  5.3.6 Measurements of 8-isoprostane  Free 8-isoprostane measurements were performed on conditioned medium from cell culture experiments, as per manufacturer's recommendations (Cayman Chemical Company, Ann Arbor, MI). Cells were treated as described in section 5.3.5 with the sole modification 152  Chapter 5 that 6-well plates were utilized in order to collect sufficient conditioned media for analysis. Also additional treatment groups including treatment with 0.9 tiM valdecoxib, and 0.75 mM aspirin were included (Cayman). Upon collection of conditioned media, 0.005% BHT (Butylated hydroxytoluene) was added to prevent further production of 8-isoprostanes in the samples. Samples were stored at -80°C until time of analysis. For free 8-isoprostane measurements, samples were measured using the EIA kit (Cayman Chemical Company, Ann Arbor, MI) as described in section 4.3.6. Briefly, samples and 8-isoprostane standards were incubated with EIA Buffer, AChE tracer and 8isoprostance antiserum on the EIA plate for 18 h at room temperature. EIA plates were developed by the addition of Ellman's reagent and 8-isoprostane tracer followed by incubation for 90 min at room temperature. Samples were measured via absorbance readings at 412 nm on the Tecan GENios Rainbow absorbance plate reader (Tecan, San Jose, CA).  5.4 Results 5.4.1 Native, adenoviral, and H/R-induced expression of CYP 2C9 in HUVECs.  Hypoxic conditions were optimized by measuring the P0 2 at varying oxygen concentrations in both the chamber air and bubbled media. Results, Figure 5.1, showed that we were able to obtain a P0 2 of approximately 10% by presetting our oxygen concentration to 1%. Measured P02 levels were comparable in the chamber air and in bubbled media indicating that our method of bubbling media was sufficient to control solution P02 levels. It should be noted that these measurements were repeated while both increasing and decreasing 153  Chapter 5  •  25  20 -  E C•1  0 10  a.  -  5• P02 atmosphere 0 P02 solution 0  00  ^  0.5^1.0^1.5^2.0^2.5^3.0  % 02 Hypoxia Chamber Figure 5.1 Relationship between hypoxic chamber oxygen concentration and measured P02. Oxygen concentrations were controlled using a hypoxic glove box. The P02 of the air and of bubbled culture media were assessed under humidified conditions at 37°C and 5% CO2. P02 was measured using Oxylab P02 probes.  154  Chapter 5  the chamber oxygen concentration and with a second P02 probe. We did not detect any variability in these measurements in our range of interest (P02 5-15). Cultured HUVECs were analyzed for native expression of CYP 2C9 by Western blot. HUVECs did not express detectable levels of CYP 2C9. HUVECs transduced with Ad-CYP 2C9 sense but not CYP 2C9 antisense expressed CYP 2C9, Figure 5.2(A). Hypoxia for 24 h followed by 4 h (H24/R4) of re-oxygenation did not induce CYP 2C9 expression in HUVECs, Figure 5.2 (B).  5.4.2 CYP 2C9 expression contributes to post H/R cell death in HUVEC  HUVECs were transduced with Ad-CYP 2C9 sense or Ad-CYP 2C9 antisense were exposed to H24/R4 or normoxic conditions (control). Cell viability was measured using the MTS assay, shown in Figure 5.3. Cells transduced with antisense demonstrated a decrease in viability following H24/R4 to 71.3 ± 0.3% of control. These results are comparable to those we routinely observe in non-transduced cells. Cells transduced with sense CYP 2C9 demonstrated a significantly greater drop in viability to 56.0 ± 1.9% of control (p<0.005 compared to antisense cells). Pre-treatment with SP did not significantly increase viability post-H24/R4 treatment in antisense transduced cells (83.0 ± 13.9% SP treated compared to 71.3 ± 0.3% untreated, p>0.1). Pre-treatment with SP did significantly increase viability post-H24/R4 treatment in sense transduced cells (77.0 ± 18.7% SP treated vs. 56.0 ± 1.9% untreated, p<0.05).  155  Chapter 5  A CYP 2C9 (56 kDa) 0-actin (43 kDa)  B wimior  Norrnoxi4  11111111110.0  CYP 2C9 (3-actin CYP 2C9  H/R  0-actin  Non-transduced  ^  Ad-CYP 2C9 sense  Figure 5.2 CYP 2C9 expression in HUVECs following adenoviral transduction and H/R.  (A) Immunoblot for CYP 2C9 in HUVECs transduced with Ad-CYP 2C9 antisense and sense. Human liver microsomes (HLM) are used as a positive control for CYP 2C9 expression. (B)Non-transduced and CYP 2C9 sense transduced cells were exposed to 24 h hypoxia followed by 4 h reperfusion and normoxic time controls. CYP 2C9 expression was analyzed by Western blot.  156  Chapter 5  0 Normoxic B H/R  Untreated 10 pM SP  ^  Untreated  Ad-CYP 2C9 antisense  ^  10 pM SP  Ad-CYP 2C9 sense  Figure 5.3 CYP 2C9 expression in HUVECs reduces cell viability following BUR.  Percent viability of HUVECs compared to control of cells exposed to 24 h hypoxia followed by 4 h reperfusion and normoxic time controls. Cells were either untreated or treated with 10 !LIM SP 1 h prior to the induction of hypoxia. Values are expressed as mean ± SE (n=6, *p<0.05).  157  Chapter 5 5.4.3 SP treatment does not alter SMC viability following H/R.  HCASMCs were pretreated with SP and exposed to H24/R4; the results are shown in Figure 5.4. H/R reduced cell viability in both untreated (86.4 ± 3.3% for H24/R4 vs. 100.0 ± 2.0% normoxic, p<0.005) and SP treated (82.3 ± 3.7% for H24/R4 vs. 94.8 ± 2.4% normoxic, p<0.005) cells. Unlike what was seen in HUVECs, SP treatment caused a slight decrease in viability in both normoxic (5.2%) and H/R exposed (4.2%) cells (p<0.05).  5.4.4 Effect of SP and COX-inhibition on 8-isoprostane production following H/R in CYP 2C9 expressing HUVECs  Levels of 8-isoprostane were measured as an indicator of reactive oxidant production. Conditioned media was collected from Ad-CYP 2C9 sense and antisense transduced cells. Cells were pre-treated with 10 p,M SP, a reversible inhibitor of CYP 2C9 inhibitor; 0.9 W\/1 valdecoxib, IC50 (half maximal inhibitory concentration) of the reversible COX-2 inhibitor; and 0.75 mM aspirin, IC50 for irreversible COX-1 inhibition. Cells were then exposed to H24/R4. 8-isoprostane levels were measured using the 8-isoprostane EIA, Figure 5.5. In the absence or presence of each inhibitor Ad-CYP 2C9 sense cells had higher levels of 8isoprostanes in the condition media than Ad-CYP 2C9 antisense cells. For example, in the absence of inhibitor 8-isoprostane levels were 33.1 ± 15.3 pg/ml for antisense cells and 86.1 ± 9.5 pg/ml for sense cells. CYP 2C9 inhibition with SP was able to reduce 8-isoprostane levels in CYP 2C9 sense transduced cells from 86.1 ± 9.5 pg/ml to 55.4 ± 4.3 pg/ml. Treatment with valdecoxib has the opposing effect with 8-isoprostane levels increasing to 112.3 ± 8.4 pg/ml. 158  Chapter 5  T) 120 ..  C  o  100  V  `15 80 ›ft  13  60  '5  40  .  O Untreated • 10 pM SP  C  G) 20  Normoxic  H/R  Figure 5.4 SP treatment in HCASMCs does not alter proliferation or cell viability following H/R. Percent viability of HCASMCs exposed to 24 h hypoxia followed by 4 h reperfusion and  normoxic time controls. Cells were either untreated or treated with 10 µM SP 1 h prior to the induction of hypoxia. Values are expressed as mean ± SE (n=6, * p<0.05).  159  Chapter 5  140 -  120  100  80  o Anti-sense n Sense  60  40  20  No Inhibitor^Sulfaphenazole^Valdecoxib  Figure 5.5 CYP 2C increases 8-isoprostane levels.  ^  Aspirin  HUVECs transduced with Ad-CYP 2C9 sense or antisense and HCASMCs were pre-treated with SP, valdecoxib or aspirin 1 h prior to induction of H/R. Cells were exposed to 24 h hypoxia in followed by 4 h of re-oxygenation in normoxic conditions. Free 8-isoprostane measurements were performed on conditioned medium by EIA. Data represents mean ± SD, n=3, * p<0.05).  160  Chapter 5 Aspirin treatment showed little effect on 8-isoprostane levels in CYP 2C9 expressing HUVECs.  5.5 Discussion Our previous studies have shown an important role for CYP 2C in post-ischemic vascular function and CAV development. (15, Chapter 4 ) In both of these studies it is hypothesized that CYP 2C induced its deleterious effects via oxidative stress on the endothelium. However, this hypothesis had not been directly tested. Thus, we examined the role of CYP 2C9 in cultured endothelial and smooth muscle cells following exposure to H/R, as an in vitro model of I/R.  CYP 2C9 is expressed in human endothelial cells. 21 However, upon culturing, CYP 2C9 levels in endothelial cells rapidly decrease both at the mRNA and protein level. 22 ' 23 Therefore, our finding that CYP 2C9 is not expressed in cultured HUVECs was not unexpected. In order to examine the role to CYP 2C9 in these cells we employed adenoviral vectors expressing sense and anti-sense (control) CYP 2C9. Previous studies have shown that CYP 2C mRNA and protein levels increase following hypoxia. 22 We examined whether exposing the cells to H/R altered CYP 2C9 levels. We were unable to detect any induction. As there are many members of the CYP 2C family it is likely that family members other than 2C9 are responsible for this induction. We have previously shown that the CYP 2C9 inhibitor SP protects against endothelium-dependent vascular dysfunction following I/R in rats.  15  Therefore, we wanted to  examine whether CYP 2C9 would also contribute to endothelial cell death and dysfunction in 161  Chapter 5 cultured endothelial cells. Our results indicate that CYP 2C9 expression results in increased cell death following exposure to H/R. Pre-treatment with SP is able to protect against H/R in HUVECs expressing CYP 2C9 sense but not anti-sense. We also found that CYP 2C9 sense expressing cells produce increased oxidant radicals, as indicated by free 8-isoprostane levels in the conditioned media, following H/R and that SP was able to reduce 8-isoprostane levels in these cells. We have previously found that SP treatment during I/R reduces subsequent SMC proliferation following heterotopic heart transplantation in rats, see Figure 4.13. Although it is likely that this resulted from decreased bioavailability of endothelium derived NO and not from the single dose of SP given prior to transplantation, we examined the possibility that SP was directly affecting SMCs. We detected only a slight decrease in the number of viable SMCs following SP treatment. H/R cause a decrease in cell viability in both untreated and SP treated groups and exposure to H/R did not increase or decrease this effect. This result agrees with our previous finding that SMC-dependent relaxation is impaired following I/R and that SP pre-treatment is not protective against this type of vascular dysfunction. 15 As described in section 1.5.4, studies related to celecoxib and paracoxib/valdecoxib have also shown an association with increased cardiovascular risks resulting in the withdrawal of paracoxib/valdecoxib from the market. 24-26 Given that increased AA liberation is associated with ischemia, it is possible that under conditions of ischemia, in the presence of coxibs, that AA liberation would stimulate elevated CYP 2C activity and subsequent ROS production. This elevation in ROS production may result in vascular injury and dysfunction that could contribute to the problems associated with coxib administration in the presence of ischemia. Therefore, we were interested in whether COX inhibition could induce increased 162  Chapter 5 oxidative damage by CYP 2C9. We hypothesized that decreased AA metabolism by COX-2 would lead to increased AA metabolism by CYP 2C9 and consequently increased oxidative stress. Our results show that valdecoxib treatment did increase 8-isoprostane formation in CYP 2C9 sense but not anti-sense expressing cells. However, aspirin, administered at a dose specific for COX-1 did not alter 8-isoprotane production in either CYP 2C9 expressing or control cells. Thus, it is not clear whether altered AA metabolism is involved. One factor complicating this analysis is that valdecoxib, like most COX-2 inhibitors, is largely metabolized via CYP 2C9. 27 Studies have not been performed to assess whether low dose valdecoxib can induce CYP 2C9 activity although high dose valdecoxib, IC50 41 ILLM, acts as a moderate inhibitor of 2C9 (Bextra label information, Pfizer Inc. NY, NY). In conclusion, we demonstrate that CYP 2C9 expression in endothelial cells leads to increased cell death and ROS production following H/R and that pre-treatment with the CYP 2C9 inhibitor SP can reduce these effects. SP did not have an effect of H/R-induced cell death in SMC. We also present intriguing data indicating that the COX-2 inhibitor valdecoxib induces increased oxidant stress in CYP 2C9 expressing endothelial cells. Further detailed experimentation will be required to elucidate the mechanism of COX-2 induced oxidant production and to determine if altered AA metabolism plays a role.  163  Chapter 5  5.6 Bibliography 1. 2.  3. 4. 5. 6. 7. 8. 9. 10. 11. 12.  13. 14.  Bieche I, Narjoz C, Asselah T, et al. Reverse transcriptase-PCR quantification of mRNA levels from cytochrome (CYP)1, CYP2 and CYP3 families in 22 different human tissues. Pharmacogenet Genomics. Sep 2007;17(9):731-742. Potente M, Michaelis UR, Fisslthaler B, et al. Cytochrome P450 2C9-induced endothelial cell proliferation involves induction of mitogen-activated protein (MAP) kinase phosphatase-1, inhibition of the c-Jun N-terminal kinase, and up-regulation of cyclin Dl. J Biol Chem. May 3 2002;277(18):15671-15676. Freyss-Beguin M, Millanvoye-van Brussel E, Duval D. Effect of oxygen deprivation on metabolism of arachidonic acid by cultures of rat heart cells. Am J Physiol. Aug 1989;257(2 Pt 2):H444-451. Leong LL, Sturm MJ, Ismail Y, et al. Plasma phospholipase A2 activity in clinical acute myocardial infarction. Clin Exp Pharmacol Physiol. Feb 1992 ;19(2): 113-118. Van der Vusse GJ, Reneman RS, van Bilsen M. Accumulation of arachidonic acid in ischemic/reperfused cardiac tissue: possible causes and consequences. Prostaglandins Leukot Essent Fatty Acids. Jul 1997;57(1):85-93. Williams SD, Gottlieb RA Inhibition of mitochondrial calcium-independent phospholipase A2 (iPLA2) attenuates mitochondrial phospholipid loss and is cardioprotective. Biochem J. Feb 15 2002;362(Pt 1):23-32. Hendrickson SC, St Louis JD, Lowe JE, et al. Free fatty acid metabolism during myocardial ischemia and reperfusion. Mol Cell Biochem. Jan 1997;166(1-2):85-94. Ogata K, Jin MB, Taniguchi M, et al. Attenuation of ischemia and reperfusion injury of canine livers by inhibition of type II phospholipase A2 with LY329722. Transplantation. Apr 27 2001;71(8):1040-1046. Sargent CA, Vesterqvist 0, McCullough JR, et al. Effect of the phospholipase A2 inhibitors quinacrine and 7,7-dimethyleicosadienoic acid in isolated globally ischemic rat hearts. J Pharmacol Exp Ther. Sep 1992;262(3):1161-1167. Luo G, Zeldin DC, Blaisdell JA, et al. Cloning and expression of murine CYP2Cs and their ability to metabolize arachidonic acid. Arch Biochem Biophys. Sep 1 1998;357(1):45-57. Fleming I, Michaelis UR, Bredenkotter D, et al. Endothelium-derived hyperpolarizing factor synthase (Cytochrome P450 2C9) is a functionally significant source of reactive oxygen species in coronary arteries. Circ Res. Jan 19 2001;88(1):44-51. Fulton D, McGiff JC, Wolin MS, et al. Evidence against a cytochrome P450-derived reactive oxygen species as the mediator of the nitric oxide-independent vasodilator effect of bradykinin in the perfused heart of the rat. J Pharmacol Exp Ther. Feb 1997;280(2):702-709. Granville DJ, Tashakkor B, Takeuchi C, et al. Reduction of ischemia and reperfusioninduced myocardial damage by cytochrome P450 inhibitors. Proc Natl Acad Sci U S A. Feb 3 2004;101(5):1321-1326. Fichtlscherer S, Dimmeler S, Breuer S, et al. Inhibition of cytochrome P450 2C9 improves endothelium-dependent, nitric oxide-mediated vasodilatation in patients with coronary artery disease. Circulation. Jan 20 2004;109(2):178-183. 164  Chapter 5 15.  Hunter AL, Bai N, Laher I, et al. Cytochrome p450 2C inhibition reduces postischemic vascular dysfunction. Vascul Pharmacol. Oct 2005;43(4):213-219. 16. Carrier M, Trudelle S, Thai P, et al. Ischemic threshold during cold blood cardioplegic arrest: monitoring with tissue pH and p02. J Cardiovasc Surg (Torino). Oct 1998;39(5):593-597. 17. Oz MC, Liao H, Naka Y, et al. Ischemia-induced interleukin-8 release after human heart transplantation. A potential role for endothelial cells. Circulation. Nov 1 1995;92(9 Suppe:11428-432. 18. Pinsky DJ, Naka Y, Liao H, et al. Hypoxia-induced exocytosis of endothelial cell Weibel-Palade bodies. A mechanism for rapid neutrophil recruitment after cardiac preservation. J Clin Invest. Jan 15 1996;97(2):493-500. 19. Wiener L, Santamore WP, Venkataswamy A, et al. Postoperative monitoring of myocardial oxygen tension: experience in 51 coronary artery bypass patients. Clin Cardiol. Aug 1982;5(8):431-435. 20. Zuurbier CJ, van Iterson M, Ince C. Functional heterogeneity of oxygen supplyconsumption ratio in the heart. Cardiovasc Res. Dec 1999;44(3):488-497. 21. Hillig T, Krustrup P, Fleming I, et al. Cytochrome P450 2C9 plays an important role in the regulation of exercise-induced skeletal muscle blood flow and oxygen uptake in humans. J Physiol. Jan 1 2003;546(Pt 1):307-314. 22. Michaelis UR, Fisslthaler B, Barbosa-Sicard E, et al. Cytochrome P450 epoxygenases 2C8 and 2C9 are implicated in hypoxia-induced endothelial cell migration and angiogenesis. J Cell Sci. Dec 1 2005;118(Pt 23):5489-5498. 23. Michaelis UR, Fisslthaler B, Medhora M, et al. Cytochrome P450 2C9-derived epoxyeicosatrienoic acids induce angiogenesis via cross-talk with the epidermal growth factor receptor (EGFR). Faseb J. Apr 2003;17(6):770-772. 24. Nussmeier NA, Whelton AA, Brown MT, et al. Complications of the COX-2 inhibitors parecoxib and valdecoxib after cardiac surgery. N Engl J Med. Mar 17 2005;352(11):1081-1091. 25. Ott E, Nussmeier NA, Duke PC, et al. Efficacy and safety of the cyclooxygenase 2 inhibitors parecoxib and valdecoxib in patients undergoing coronary artery bypass surgery. J Thorac Cardiovasc Surg. Jun 2003;125(6):1481-1492. 26. Solomon SD, McMurray JJ, Pfeffer MA, et al. Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma prevention. N Engl J Med. Mar 17 2005;352(11):1071-1080. 27.^Ibrahim A, Park S, Feldman J, et al. Effects of parecoxib, a parenteral COX-2specific inhibitor, on the pharmacokinetics and pharmacodynamics of propofol. Anesthesiology. Jan 2002;96(1):88-95.  165  Chapter 6: Summary and Conclusions  6.1 Restatement of the Problem More than 3,000 heart transplants are performed annually worldwide. Current immunosuppressive regimens are very effective in preventing acute rejection. However, chronic rejection associated with CAV remains the major hurdle to long-term graft survival of all vascularized organ transplants. CAV is an accelerated and diffuse form of arteriosclerosis' that can be detected in up to 75% of heart transplant recipients following the first year of transplantation. 2 Although immunological mechanisms clearly play an important role in the pathogenesis of CAV, non-immunological mechanisms, such as peri-transplant  UR injury, also contribute via direct damage or indirectly through cross-talk with immune responses associated with this type of vasculopathy. 3 ' 4 There are relatively few treatments for established CAV and re-transplantation is often the only option. Given the short supply of donor organs and the added stress of an additional major surgery, even re-transplantation is not a viable option for many patients. Treatments to control risk factors, such as CMV infection s and hyperlipidemia 6 , have shown the most promising results at preventing CAV development. Given the association between vascular I/R injury and CAV development our primary aim was to examine whether inhibition of peri-transplant FR injury could reduce or prevent CAV treatment. We selected two targets that are known to protect against and contribute to oxidant injury, ARC and the  166  Chapter 6 CYP 2C enzymes, respectively, to examine this link based on results shown in myocardial infarction?' 8  6.2 Summary of Findings Initially, we examined the ability to ARC to protect against oxidant stress in vascular cell types (Chapter 2). We demonstrated, for the first time, that ARC is expressed in both cultured endothelial and smooth muscle cells by RT-PCR and immunoblotting. We then increased ARC levels utilizing TAT-fusion protein transduction and examined ARC's ability to protect against oxidant-mediated cell death induced by treatment with H202. TAT-ARC did not confer increased protection against H202 treatment than did treatment with our control protein TAT-0-gal. These results differed from results obtained in H9c2 rat embryonic cardiomyocytes. 8 During our control experiments analyzing the action of ARC in H9c2 cells, we observed that ARC overexpressing cells did not undergo differentiation induced by serum withdrawal. We examined this observation further and demonstrated that ARC expression levels increase and stabilize upon differentiation in non-transduced H9c2 cells. ARCoverexpression in pre-differentiated H9c2-cells suppressed differentiation; indicated by increased myotube formation, nuclear fusion and expression of the differentiation markers myogenin and troponin-T. ARC-overexpression inhibited myoblast differentiation associated caspase-3 activation, suggesting ARC inhibits myogenic differentiation through caspase inhibition. Thus, we have demonstrated a novel role for ARC in the regulation of muscle differentiation. 167  Chapter 6 As we were unable to obtain sufficient preliminary data indicating a role for ARC in protection against oxidative damage in the vasculature, we turned our attention to the CYP 2C family of enzymes. Specifically, we examined the role of the CYP 2C9-like enzyme in rodents. Our initial examinations involved assessment of vascular function following ischemia and reperfusion (Chapter 3). Previous studies have shown that vascular function is impaired following ischemic injury 9-14 and that CYP inhibitors provide protection against myocardial infarction 7 and vascular dysfunction in patients with manifest coronary artery disease. 15 Therefore, we hypothesized that SP, an inhibitor of CYP 2C9, would also attenuate post-ischemic endothelial dysfunction. We utilized the Langendorff model of I/R in rats and analyzed vascular function in septal coronary resistance arteries by pressure myography. 16 I/R caused impairment in both endothelium-dependent and independent vasodilation. Pretreatment with SP restored endothelial sensitivity to ACh but did not restore sensitivity to endothelium-independent vasodilators. I/R-induced superoxide production was assessed by dihydroethidium staining of flash frozen hearts. SP treatment significantly reduced superoxide production in arterial walls following I/R injury. Therefore, we concluded that CYP 2C contributes to impaired post-ischemic endothelium-dependent, NO-mediated vasodilation by increasing superoxide production. Given the protective role of the CYP 2C inhibitor SP in protection against vascular dysfunction following I/R and the link between peri-transplant I/R injury, post-transplant vascular dysfunction and CAV, we explored whether CYP 2C may also contribute to the onset of CAV (Chapter 4). Lewis-to-Fisher rat heterotopic heart transplants were performed. Donors and recipients were treated with 5 mg/kg SP or vehicle control 1 h prior to surgery. We were able to demonstrate that SP did not affect post-transplant morbidity, mortality or  168  Chapter 6 weight gain. Assessment of coronary blood vessels from rats 30 days post-transplant indicated that treatment with SP significantly reduced luminal narrowing. However, SP did not reduce diffuse, focal, epicardial, endocardial or perivascular immune infiltration nor did it alter infiltration by lymphocytes as measured by CD3 + and CD8 + staining. SP also did not significantly alter TUNEL positivity in myocardial, endothelial or SMC populations. We did not observe endothelial loss in either the SP-treated or control groups. Analysis of rats 4 days post-transplant demonstrated a decrease in SMC proliferation in the SP-treated rats compared to controls. In addition, the SP-treatment group had decreased levels of serum IFN-y and 8soprostane post-transplantation. Our final set of experiments examined the effects of CYP 2C9 in cultured vascular cells in response to H/R. We demonstrated that HUVECs do not express CYP 2C9 following culture and that H/R does not induce expression. We successfully induced expression of CYP 2C9 through the use of adenoviral constructs and demonstrated that CYP 2C9 contributes to cell death and oxidant stress following H/R in these cells. CYP 2C9 inhibition, by SP, reduced these effects. In contrast, SP treatment had no effect on SMC viability following H/R. In addition, we examined the potential for COX inhibitors to alter CYP 2C9 production of ROS. Our results indicated that the COX-2 inhibitor, valdecoxib, but not aspirin given at a dose specific for COX-1, caused increased ROS production in CYP 2C9 expressing cells. It is unclear if valdecoxib is exerting this effect through direct induction of CYP 2C9, altered AA metabolism or an alternative pathway.  169  Chapter 6  6.3 Relevance of Findings The relationship between peri-transplant ischemic injury and CAV has been previously described. 3 ' 4. 17-21 However, effective strategies for CAV by preventing ROS production during peri-transplant I/R have not been reported. Some previous studies have linked peri-operative ROS scavenging treatments using antioxidants which reduced CAV development. Murata et al. 22 demonstrated that perioperative treatment with the SOD mimetic m40401 reduced CAV development. More recently, Iwanaga et a1. 23 demonstrated that peri-operative treatment with the antioxidant riboflavin reduces both acute rejection and CAV development in mice. Complementary studies have been described in renal transplantation where the use of peri-operative antioxidants have reduced both acute and chronic obliterative arteriosclerosis. 24 ' 25 Studies targeting the sources of ROS have been examined in the field of myocardial I/R related to infarction. As described in section 1.2.2, several candidate pathways have been proposed to produce ROS during UR. These include mitochondria, NADPH oxidases, xanthine oxidase and eNOS. However, attempts to target these systems in I/R injury have not met great success. NADPH oxidase inhibition, by use of p47-null mice, revealed no significant difference in infarct size. 26 Xanthine oxidase inhibitors have also failed to protect against I/R and may be contraindicated in patients with ischemic disease 26' 27 while eNOS may play a protective role. 28 Thus, the discovery that CYP 2C9 inhibition significantly reduced myocardial infarction was exciting. 7 Our studies, targeting ROS production by CYP 2C9, are the first, to our knowledge, to significantly reduce both peri-transplant vascular dysfunction and CAV. Our method of 170  Chapter 6 administering SP, to inhibit CYP 2C9, only during the peri-operative period demonstrated a clear link between peri-operative I/R and the development of vascular dysfunction and CAV.  6.4 Future Directions Throughout the course of this thesis we have made several interesting and novel findings. Each of these findings warrants further examination. With respect to our findings related to ARC in the vasculature, we were not able to show protection against oxidative injury induce by f202. However, as ARC is expressed in the vasculature, it is logical to assume that it is serving a function in these vascular beds and ARC has been found to be a multifactorial anti-apoptotic protein.  29  Thus there are many  potential targets for ARC in these cells. Further experimentation examining alternative inducers of apoptosis and necrosis, both oxidative and non-oxidative may uncover a role for ARC in these cells. Our findings related to ARC's inhibition of myocyte differentiation indicate that the mechanism likely involves inhibition of caspase-3 activity. However, further experimentation would also be required to fully elucidate the detailed mechanism of action. Our studies of CYP 2C, related to vascular cell death and dysfunction and CAV, also create many interesting questions. If our hypothesis is correct and SP treatment protects against vascular injury by increasing post-ischemic NO bioavailability, then it is not clear why direct addition of NO•, by the NO donor SNP, did not have a similar effect. This effect was also described by Fichtlsherer  et al.. 15  It is possible that this difference reflects the  mechanism of NO- transfer between endothelial and SMCs. It is also possible that SP's 171  Chapter 6 restoration of endothelium-dependent vasodilation involves other factors outside of maintenance of NO bioavailability, likely related to decreased oxidative damage to the endothelium. Direct examination of the role of AA release and examination of alternative CYP 2C substrates would undoubtedly provide mechanistic insight into CYP 2C role in I/R injury. Experiments related to detailed examination of alterations in AA metabolism have commenced in our laboratory. These experiments would not only provide insight into CYP 2C deleterious effects during I/R but may also provide insight into why COX-2 inhibitors have been associated with increased risk of cardiovascular events. Our finding that CYP 2C inhibition during the peri-transplant period could have clinical importance. In order to translate this research into a clinical setting there are several questions that should be addressed. One intriguing possibility is that addition of CYP 2C inhibitors to cardioplegic solutions could be sufficient to confer protection. This situation would be advantageous as it would not require pre-treatment of donors and would likely reduce unwanted drug-drug interactions with cardiovascular drugs necessary for recipient treatment. If donor and recipient treatment are required then definning the time-line for inhibition would be required. Also, dosing considerations for all therapeutic drugs that are metabolised by the CYP 2C family would also have to be considered.  6.5 Concluding Remarks  Our overarching goal of these studies was to examine methods of preventing or reducing vascular I/R injury and subsequent development of CAV. Although we did not obtain  172  Chapter 6 positive results in our preliminary data related to ARC in vascular oxidative damage we did uncover a serendipitous role for ARC in myogenic differentiation. In our studies related to CYP 2C9, we were able to reduced post-ischemic oxidative stress, reduce endotheliumdependent vascular dysfunction and significantly reduce CAV development.  173  Chapter 6  6.6 Bibliography  1. 2.  3. 4. 5.  6. 7. 8. 9. 10. 11. 12. 13. 14.  Dong C, Granville DJ, Tuffnel CE, et al. Bax and apoptosis in acute and chronic rejection of rat cardiac allografts. Lab Invest. Dec 1999; 79(12): 1643-1653. Yeung AC, Davis SF, Hauptman PJ, et al. Incidence and progression of transplant coronary artery disease over 1 year: results of a multicenter trial with use of intravascular ultrasound. Multicenter Intravascular Ultrasound Transplant Study Group. J Heart Lung Transplant. Nov-Dec 1995;14(6 Pt 2):S215-220. Wilhelm MJ, Kusaka M, Pratschke J, et al. Chronic rejection: increasing evidence for the importance of allogen-independent factors. Transplant Proc. Aug 1998;30(5):2402-2406. Laskowski I, Pratschke J, Wilhelm MJ, et al. Molecular and cellular events associated with i schemia/reperfu si on injury. Ann Transplant. 2000 ;5 (4): 29-35 . Valantine HA, Luikart H, Doyle R, et al. Impact of cytomegalovirus hyperimmune globulin on outcome after cardiothoracic transplantation: a comparative study of combined prophylaxis with CMV hyperimmune globulin plus ganciclovir versus ganciclovir alone. Transplantation. Nov 27 2001;72(10):1647-1652. Wenke K, Meiser B, Thiery J, et al. Impact of simvastatin therapy after heart transplantation an 11-year prospective evaluation. Herz. Aug 2005;30(5):431-432. Granville DJ, Tashakkor B, Takeuchi C, et al. Reduction of ischemia and reperfusioninduced myocardial damage by cytochrome P450 inhibitors. Proc Nall Acad Sci U S A. Feb 3 2004;101(5):1321-1326. Gustafsson AB, Sayen MR, Williams SD, et al. TAT protein transduction into isolated perfused hearts: TAT-apoptosis repressor with caspase recruitment domain is cardioprotective. Circulation. Aug 6 2002;106(6):735-739. Benvenuti C, Aptecar E, Mazzucotelli JP, et al. Coronary artery response to coldpressor test is impaired early after operation in heart transplant recipients. J Am Coll Cardiol. Aug 1995;26(2):446-451. Legare JF, Issekutz T, Lee TD, et al. CD8+ T lymphocytes mediate destruction of the vascular media in a model of chronic rejection. Am J Pathol. Sep 2000;157(3):859865. Moien-Afshari F, McManus BM, Laher I. Immunosuppression and transplant vascular disease: benefits and adverse effects. Pharmacol Ther. Nov 2003;100(2):141-156. Mugge A, Heublein B, Kuhn M, et al. Impaired coronary dilator responses to substance P and impaired flow-dependent dilator responses in heart transplant patients with graft vasculopathy. J Am Coll Cardiol. Jan 1993;21(1):163-170. Vassalli G, Gallino A, Kiowski W, et al. Reduced coronary flow reserve during exercise in cardiac transplant recipients. Circulation. Feb 4 1997;95(3):607-613. Weis M, Wildhirt SM, Schulze C, et al. Coronary vasomotor dysfunction in the cardiac allograft: impact of different immunosuppressive regimens. J Cardiovasc Pharmacol. Dec 2000;36(6):776-784.  174  Chapter 6 15.  Fichtlscherer S, Dimmeler S, Breuer S, et al. Inhibition of cytochrome P450 2C9 improves endothelium-dependent, nitric oxide-mediated vasodilatation in patients with coronary artery disease. Circulation. Jan 20 2004;109(2):178-183. 16. Hunter AL, Bai N, Laher I, et al. Cytochrome p450 2C inhibition reduces postischemic vascular dysfunction. Vascul Pharmacol. Oct 2005 ;43(4): 213-219. Land W, Messmer K. The impact of ischemia/reperfusion injury on specific and non17. specific early and late chronic events after organ transplantation. Transplantation Rev. April 1996;10(2):108-127. 18. Day JD, Rayburn BK, Gaudin PB, et al. Cardiac allograft vasculopathy: the central pathogenetic role of ischemia-induced endothelial cell injury. J Heart Lung Transplant. Nov-Dec 1995;14(6 Pt 2):S142-149. 19. Valantine HA. Cardiac allograft vasculopathy: central role of endothelial injury leading to transplant "atheroma". Transplantation. Sep 27 2003;76(6):891-899. 20. Tilney NL, Paz D, Ames J, et al. Ischemia-reperfusion injury. Transplant Proc. FebMar 2001;33(1-2):843-844. 21. Waaga AM, Gasser M, Laskowski I, et al. Mechanisms of chronic rejection. Curr Opin Immunol. Oct 2000;12(5):517-521. 22. Murata S, Miniati DN, Kown MH, et al. Superoxide dismutase mimetic m40401 reduces ischemia-reperfusion injury and graft coronary artery disease in rodent cardiac allografts. Transplantation. Oct 27 2004;78(8):1166-1171. 23. Iwanaga K, Hasegawa T, Hultquist DE, et al. Riboflavin-mediated reduction of oxidant injury, rejection, and vasculopathy after cardiac allotransplantation. Transplantation. Mar 27 2007;83(6):747-753. 24. Land W, Schneeberger H, Schleibner S, et al. The beneficial effect of human recombinant superoxide dismutase on acute and chronic rejection events in recipients of cadaveric renal transplants. Transplantation. Jan 1994;57(2):211-217. 25. Vela C, Cristol JP, Maggi MF, et al. Oxidative stress in renal transplant recipients with chronic rejection: rationale for antioxidant supplementation. Transplant Proc. Feb-Mar 1999;31(1-2):1310-1311. 26. Hoffmeyer MR, Jones SP, Ross CR, et al. Myocardial ischemia/reperfusion injury in NADPH oxidase-deficient mice. Circ Res. Oct 27 2000;87(9):812-817. 27. Parmley LF, Mufti AG, Downey JM. Allopurinol therapy of ischemic heart disease with infarct extension. Can J Cardiol. Apr 1992;8(3):280-286. 28. Sharp BR, Jones SP, Rimmer DM, et al. Differential response to myocardial reperfusion injury in eNOS-deficient mice. Am J Physiol Heart Circ Physiol. Jun 2002;282(6):H2422-2426. 29.^Ekhterae D, Lin Z, Lundberg MS, et al. ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis in heart-derived H9c2 cells. Circ Res. Dec 9 1999;85(12):e70-77.  175  Appendix I  Appendix I: Animal Care Certificate for Transplantation  ^ 1*  https://rise.ubc.ca/rise/Doc/0/H7H8J5M9E6LK3BO1 . I t34AOQFLAAL.  THE UNIVERSITY OF BRITISH COLUMBIA  ANIMAL CARE CERTIFICATE Application Number: A05-0019 Investigator or Course Director: David J. Granville Department: Pathology & Laboratory Medicine Animals:  Start Date:  Rats F344 112 Rats Lewis 205  January 2, 2005  Approval Date:  March 22, 2006  Funding Sources: Grant Agency: Grant Title:  Canadian Institutes of Health Research The role of CYP2C9 in peri-transplant ischemic injury and transplant vascular disease  Grant Agency: Grant Title:  Dean of Medicine Start Up Funding  Grant Agency:  Heart and Stroke Foundation of B.C. & Yukon The role of CYP2C9 in peri-transplant ischemic injury and transplant vascular disease  Grant Title: Grant Agency: Grant Title:  Heart & Stroke Foundation of Canada Role of CYP2C9 in Transplant Vascular Disease  Grant Agency: Grant Title:  Canadian Institutes of Health Research Role of CYP2C9 in Cardiac Ischemia and Reperfusion Injury  Grant Agency:  St. Paul's Hospital Cytochrome p450 monooxygenases: Role in endothelial and smooth muscle cell death and cardiac transplant vascular disease  Grant Title:  1 of 2^  11%29/2007 6:33 PM  176  Appendix I  https://rise.ubc.ca/rise/Doc/0/1 -17H8J5M9E6LK3BOFTB4AOQFLAA , ...  Grant Agency: Michael Smith Foundation for Health Research Grant Title:^Transplantation training program  Unfunded title: CYP2C9 contributes to cardiac ischemia and reperfusion injury The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies.  A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093  2 of 2  ^  11/29/2007 6:33 PM  177  Appendix II  Appendix II: Rat Heterotopic Heart Transplantation SOP SOP: The technique for heterotopic cardiac transplantation in rats Submitted by: Alexandra Kerjner Date: 05/05/2006  The technique for heterotopic cardiac transplantation in rats Purpose:  To describe a technique for performing heterotopic cardiac grafting in a rat model. Policy:  The personnel conducting the surgery must have received formal training by a senior staff member proficient in the technique or have equivalent previous experience. Responsibility:  Veterinarian, Technical Personnel, Investigator Materials: Micro Instruments  • • •  1 curved microneedle driver 1 straight microneedle driver 1 straight microscissor 1 45° angle microscissor 3-4 Jeweller's forceps 1 angled Jeweller's forceps  Macro Instruments  • 4 haemostats (1 large, 2 medium, 1 small) • 1 small curved Steven scissor • 1 suture scissor • 1 large scissor • 2 large forceps 178  Appendix II •  • •  1 large needle driver  1 Allis forceps 1 large curved paediatric clamp  The procedure is carried out using an operating microscope at a magnification between 4 to 25X. Procedure: In this model the donor ascending aorta is sutured end-to-side to the recipient abdominal aorta and the donor pulmonary artery is anastomosed to the recipient inferior vena cava (IVC). Hearts transplanted heterotopically behave functionally as aorto-caval fistulae. Blood enters the donor, ascending aorta from the recipient abdominal aorta and is diverted into the coronary arteries by the closed aortic valve. After the myocardium is perfused, venous blood drains into the right atrium through the coronary sinus and is pumped back into the recipient NC by the right ventricle. Recipient^Recipient (VC^abdominal aorta  / Donor ascending aorta  Donor pulmonary artery  Donor operation:  Drawing of the anastomoses in the heterotopic cardiac grafts. The donor ascending aorta is sutured end-to-side to the recipient abdominal aorta and the donor pulmonary artery is anastomosed to the recipient inferior vena cava (IVC).  The rat is anaesthetized with Xylazine (10 mg/kg)/Ketamine (120 mg/kg), IP. The rat is shaved, prepped with alcohol and betadine and moved to the surgery table. Sterile drapes are placed; aseptic technique being used throughout. The donor's abdominal cavity is opened with a large longitudinal incision long the abdomen, at the bottom of the ribs. The diaphragm and the lateral aspects of the rib cage are cut and the abdominal contexts are moved to the left side, exposing the IVC. The IVC is isolated above the liver and a loose tie is placed proximally, the NC is clamped distally. Using a 3m1 syringe and a 25 gauge needle, the NC is cannulated and slowly perfused with heparinized saline, making sure that no air gets into 179  Appendix II the vessel. The right and left superior vena cava (SVC) are isolated and two ligatures are placed leaving space between then. The SVC is cut between the two ligatures. The ascending aorta is cut below the brachiocephalic artery and the main pulmonary artery is cut proximal to its bifurcation, they are flushed with heparinized saline. The connective tissue between the ascending aorta and pulmonary artery is cut away at this stage. The pulmonary veins are ligated together with a single 6-0 silk tie. The donor heart is gently detached and flushed with heparinized saline. At this point it might be necessary to tie off a small vessel very close to the aorta and pulmonary artery. The heart is then weighed and placed into a heparinized saline bowl on ice. Superior vena caN a (3)  Inferior vena ea . (. I .) ,  Donor preparation. Superior and inferior vena cava, azygos vein and pulmonary vein were ligated. Ascending and pulmonary artery were cut in this order. Recipient preparation:  The recipient is prepared for grafting prior to procedure of the donor organ in order to reduce the ischemic times. The animal is weighed, anaesthetized with isoflurane inhalation anaesthesia in an induction chamber set at 4% anaesthesia and prepped in the same manner as the donor. Ophthalmic ointment (Lacri-lube) is put in the eyes to prevent drying and the animal is ear marked. A well insulated heating pad is positioned under the rat so that the 180  Appendix II body temperature is maintained at 38°C. A midline incision is made down to the pubic region. The bowel is brought out and wrapped in warm moist gauze. The abdominal wall is retracted on either side using a needle and silk to get good exposure of the aorta, inferior vena cava, and left-kidney vessels. The IVC and abdominal aorta are cleaned and appropriate sites for anastomoses are located. Haemostat is applied to both vessels below the level of the kidneys. The donor heart is then removed from the ice and placed in the right flank of the rat. After ensuring that the orientation of the donor PA is correct, end-to-side anastomosis between donor PA and recipient IVC is performed using continuous 9-0 nylon sutures. The posterior wall is sutured within the vessel lumen without repositioning the graft. The anterior wall is then closed externally using the same suture. Once the venous anastomosis is completed, the vein is gently stretched before tying the sutures to avoid narrowing at the anastomotic site. Arterial anastomosis: The arterial anastomosis between the donor aortic cuff and recipient aorta is performed in the fashion as the venous anastomosis. A small quantity of microfibrillar collagen (Avitene) is placed around the arterial anastomosis before releasing the clamp. Gentle pressure is applied to the anastomotic site with a dry cotton swab for 1-2 minutes after the clamp is removed. Sites of possible leaks are checked. The bowel is returned to the abdominal cavity. The abdomen and skin are closed in a two layer closure, muscle then skin. The rat receives 10 cc of saline once the incision is closed. The rat is placed in the paediatric incubator for a few hours with supplemental oxygen. Buprenophrine is administered subcutaneously at 0.01 mg/kg immediately following surgery. If necessary additional buprenophrine will be administered twice every 8-12 hours given signs of postoperative pain. Signs of postoperative pain include: decreased activity or a reluctance to move, abnormal posture or gait (i.e., arched back or lameness), rough, greasy-looking coat (due to lack of normal grooming), dark red porphyrin staining around the eyes and nose in rats (Chromodacryorrhoea), unusual aggressiveness when handled.  181  Appendix H  Scoring System for Heart Transplant Animals This system is designed to give an overview of the health status of experimental mice and rats involved in heart transplant projects. These animals undergo the very strenuous and stressful surgical procedure of having a heart transplanted into their abdomen. Each animal is monitored very carefully and scored daily using this system. The animal unit's staff is always available to give assistance and advice. Please do not hesitate to contact us if you have any questions or concerns.  Animals are evaluated on a number of criteria and given a score from 0 to 4.  Score from: 0=normal 1= minimal/mild but noticeable 4= moderate to severe  If the score is 4 for any criteria, consult with the animal unit group and/or designated experiment representative to have a second scoring done by that person. If the score is still 4, appropriate steps for treatment or euthanasia may need to be taken.  If the combined score for all of the criteria is 10 or higher, consult with the animal unit group and/or designated experiment representative regarding the appropriate action to be taken (i.e. euthanasia or treatment). A score of 10 of higher indicates a significant problem and will require intervention.  NOTE: If treatment or intervention of any kind will render the experiment unusable please inform animal unit staff at the onset of the experiment. If this is the case, animals which display illness or other problems will be euthanized.  Attitude: 0= BAR (bright, alert/active and responsive) 1= Burrowing or hiding, quiet but rouses when touched  182  Appendix II 4= No cage exploration when lid is off, burrows/hides, head presses, may or may not vocalize or be unusually aggressive when touched, no nesting, may seem confused/irritated or hyper responsive.  Appearance: 0= normal 1= mild piloerection, mild to moderate dehydration, soft stool 4= severe piloerection, moderate to sever dehydration (obvious at first glance), sunken/wasted appearance, diarrhoea (moderate to severe can be smelled easily and seen on light coat colors easily), laboured breathing, yellowing or whitish looking mucus membranes (skin) colour, animal is hot or cold to the tough.  Gait/Posture: 0= normal 1= mild in coordination when stimulated, slight hunched posture, slight limp 4= obvious ataxia or head tilt, severe hunching, tippy-toeing, favouring of limb/noticeable limp or paralysis of limb(s) Weight: (post surgical or post experiment) 0= none or up to 10% weight loss 1= 11-15% weight loss 4= 16-20% weight loss  Appetite: 0= normal, eats dry food, evidence of urine and feces on cage bottom, food missing from hopper or floor, Jell-O or supplements gone after 8 hours 1= no evidence of eating dry food but likes Jell-O or supplements 4= no interest in food or supplements  183  Appendix II In addition to the categories which are given a score, here is a list of things which must also be evaluated on a regular basis. If any of these are noted, please consult with animal unit staff or designated experiments representative for advice on appropriate action to be taken.  • • • •  Suture dehisce (incision comes open) Check incision/experimental site daily Skin lesions/sores appear Porphyrin staining in rats can be none to mild staining around eyes or nostrils (face) but if heavy or noted on pays may indicate a problem. • Fighting/ scabbing noted or excessive barbering with sores. (If barbering without sores is seen, note on cage card but you do not need to inform animal unit staff) • Weigh experimental animal daily • Check own animals (minimum) once daily or as often as required depending on experiment and reactions. Have others check your experimental animals occasionally to minimize bias. *** If any treatments are indicated they must be approved beforehand by the PI in order to assure the treatment will not interfere in any way with the experiment.  Heart Transplant Specific Items: There are daily observations needed specific to heart transplant project and must be made each time the animal is evaluated. Animals are evaluated daily beginning 2 days prior to transplant surgery and continuing for the duration of the animal's participation in the experiment. • Transplant heart palpation a) Palpate and score the quality of the heart beat. Record with observations. Heartbeat is graded as A (strong heartbeat), B (weak heartbeat), or C (no heartbeat felt). b) If the heart is no longer beating before the animal reached day 7, the data obtained will not be considered useful and the animal should be euthanized. Contact Dr. Kerjner as tissues may be needed. If Dr. Kerjner is unavailable, euthanize the animal and collect the native and transplanted hearts and placed in separate containers of formalin. Notify Dr. Kerjner when she is available • Hind limp paralysis a) If one leg is paralyzed, record observations daily and euthanize if paralysis persists longer than 1 week. b) If both legs are paralyzed, euthanize animals immediately. Notify Dr. Kerjner and collect native and transplanted hearts in formalin • Weigh animals at each evaluation and record with observations. 184  Appendix II • Animals are kept for 4, 7 or 30 days post surgery and treated daily with immunosuppressive drugs for 14 to 30 days. Make sure to record injections and any treatments or manipulations in daily record sheets. This scoring/evaluation system was taken and adapted from the Animal Care and Use Guidelines of the University of Florida.  185  Appendix III  Appendix III: List of Publications, Abstract, Oral Presentation and Awards Published Refereed Papers 1. Hunter AL, Kerjner A, Mueller KJ, McManus BM and Granville DJ. (2008).  2. 3.  4.  5. 6.  7.  8.  Cytochrome p450 2C enzymes contribute to peri-transplant ischemic injury and cardiac allograft vasculopathy. Am J Transplant. (In revision with invitation to resubmit, January 2008) Elmi S, Sallam NA, Rahman MM, Teng X, Hunter AL, Moien-Afshari F, Khazaei M, Granville DJ, Laher I. (2008) Sulfaphenazole treatment restores endothelium-dependent vasodilation in diabetic mice. Vascul Pharmacol. 48(1):1-8. Hunter AL, Zhang J, Chen SC, Si X, Wong B, Ekhterae D, McManus BM, Luo fl, Granville DJ. (2007). Prevention of myocyte differentiation by apoptosis repressor with caspase recruitment domain (ARC). FEBS Lett. 581(5):879-84. Hunter AL*, Bai N*, Laher I, Granville DJ. (2005). Cytochrome p450 2C inhibition reduces post-ischemic vascular dysfunction. Vascul Pharmacol. 43: 213-219. Li G, Chen N, Roper RL, Feng Z, Hunter AL, Danilla M, Upton C, Buller RML. (2005). Complete coding sequences of the rabbitpox virus genome. J Gen Virol. 86:2969-77 Hunter AL*, Cruz RP*, Cheyne BM, McManus BM, Granville DJ. Cytochrome p450 Enzymes and Cardiovascular Disease. (2004). Can J of Physiol and Pharmacol. 82: 1053-60 Choy JC, Hung VHY, Hunter AL, Cheung PK, Luo Z, Motyka B, Goping IS, Sawchuck T, Bleackley RC, Podor, TJ, McManus BM, and Granville DJ. (2004). Granzyme B induces smooth muscle cell death in the absence of perforin: implications for the proteolysis of extracellular proteins. Arteriosd Thromb Vasc Biol 24(12):2245-50. Upton C, Slack S, Hunter AL, Ehlers A, Roper RL. (2003). Poxvirus orthologous clusters: toward defining the minimum essential poxvirus genome. J Virol. 77:7590-600.  Book Chapters 1. Hunter AL*, Choy JC*, Granville DJ. (2005). Detection of apoptosis in cardiovascular diseases, in Molecular Cardiology: Methods and Protocols (Sun Z. ed.). Humana, Totowa, NJ. Vol. 112:277-89  186  Appendix III  Published Abstracts 1. Hunter AL, Kerjner A, Mueller KJ, McManus BM, Granville DJ (2007) Cytochrome  p450 (CYP) 2C Contributes to Cardiac Allograft Vasculopathy. ISHLT 27th Annual Meeting and Scientific Sessions, April 25 — 28, in San Francisco. 2. Hunter AL, Chehal M, McManus BM, Granville DJ (2006) Cytochrome p450 2C Increases endothelial dysfunction following ischemia and reperfusion. The 3 rd Annual National Research Forum for Young Investigators in Cardiovascular and Respiratory Health, Winnipeg, MB. Exp Clin Cardiol. 11:1, 50. 3. Hunter AL, Bai N, McManus BM, Laher I, Granville DJ. (2005) Inhibition of Cytochrome p4-50 2C Restores Vascular Function Following Global Ischemia and Reperfusion. Canadian Cardiovascular Congress 2005, Montreal QC. Canadian Journal of Cardiology Vol 21(C). 4. Hunter AL, Bai N, McManus BM, Laher I, Granville DJ. (2005). Sulfaphenazole reduces superoxide generation and improves vascular function following ischemia and reperfusion. Experimental Biology 2005 meeting in San Diego, CA, April 4, 2005. The FASEB Journal. 19(4): A485. 5. Hunter AL Bai N, McManus BM, Laher I, Granville DJ. (2005) Cytochrome p450 enzymes contribute to superoxide production and vascular dysfunction following ischemia and reperfusion. The 2 nd Annual National Research Forum for Young Investigators in Cardiovascular and Respiratory Health, Winnipeg, MB. Exp Clin Cardiol. 10:1, 31. 6. Hunter AL, Chen YL, Chen SC, Gustafsson AB, Gottlieb RA, McManus BM, Granville DJ. (2004). Role of Apoptosis Repressor with Caspase Recruitment Domain (ARC) in Endothelial Cell Death. The 1 st Annual National Research Forum for Young Investigators in Cardiovascular and Respiratory Health, Winnipeg, MB. Exp Clin Cardiol. 9:56. 7. Hunter AL, Chen YL, Chen SC, Gustafsson AB, Gottlieb RA, McManus BM, Granville DJ. (2004). TAT-Mediated Protein Transduction of Apoptosis Repressor with Caspase Recruitment Domain (ARC) in Endothelial and Smooth Muscle Cells. The 93rd Annual United States and Canadian Academy of Pathology Meeting. Vancouver, BC. Modern Pathology. 17(Supp 1): 57A 8. Granville DJ, Choy JC, Hunter AL, Kerjner A, Goping IS, Sawchick T, Jirik FR, Bleackley C, McManus BM. (2003). Granzyme B-mediated smooth muscle cell apoptosis contributes to medial degeneration in cardiac allograft vasculopathy. American Heart Assoc Scientific Conference on Molecular Mechanisms of Growth, Death and Regeneration in the Myocardium, Snowbird, UT. 9. Roper RL, Li G, Chen N, Hunter AL, Buller RML, and Upton C. (2003). Complete Rabbitpox Virus Genome Sequence, Phylogeny and Virulence Factors. 22nd Annual American Society for Virology Meeting, Davis, California.  187  Appendix III Oral Presentations  Invited presentations 1. "Cytochrome p450 2C Contributes to Ischemia and Reperfusion Injury and Cardiac Allograft Vasculopathy" (2007). Center for Cardiovascular Biology and Regenerative Medicine, University of Washington Medicine, Department of Pathology, Seattle, WA. 2. "Cytochrome p450 2C Contributes to Post-Ischemic Vascular Dysfunction and Cardiac Allograft Vasculopathy" (2006). Heart Transplant Research Group at the Alberta Stollery Children's Hospital, Edmonton, AB.  Podium presentations 1. "Cytochrome p450 (CYP) 2C Contributes to Cardiac Allograft Vasculopathy." (2007). ISHLT 27th Annual Meeting and Scientific Sessions, in San Francisco. 2. "Cytochrome p450 2C Contributes to Post-Ischemic Vascular Dysfunction and Cardiac Allograft Vasculopathy" (2006). Centre for Blood Research/ IMPACT Research Day. Vancouver, BC. (Best overall oral presentation, $150). 3. "Inhibition of Cytochrome p450 2C Restores Vascular Function Following Global Ischemia and Reperfusion". (2005). Canadian Cardiovascular Congress 2005, Montreal QC.(l st prize oral presentation from Canadian Society for Atherosclerosis, Thrombosis and Vascular Biology, $500.) 4. "Cytochrome p450 2C9 Inhibition Reduces Post-ischemic Superoxide and Vascular Dysfunction." (2005) Pathology Research Day 2005, University of British Columbia. (Outstanding oral presentation award, $300). 5. "Regulation of Cell Death by Apoptosis Repressor with Caspase Recruitment Domain (ARC) in Endothelial and Smooth Muscle Cells". (2003). 7 th Annual BC Transplantation Research Day, Vancouver BC. (1st prize oral presentation, $500). Awards 1. 2. 3. 4.  Heart and Stroke Foundation of Canada, Doctoral Research Award, $62,000, 06/06-06/09 Michael Smith Foundation (MSFHR), Junior Trainee Award, $45,000, 09/04-09/06 MSFHR, Senior Trainee Award, $45,000 (partially declined), 06/04-08/08 Canadian Society for Atherosclerosis, Thrombosis and Vascular Biology (CSATVB), 2005 Top Oral Presentation Award, $500 5. Canadian Cardiovascular Society, 'Have a Heart Bursary', approx $2,500, 10/05 6. CSATVB, Travel Award, $1,500, 05/05 7. Canadian Society of Transplantation, Travel Award, $1,500, 05/04 8. Centre for Blood Research/IMPACT, Best Oral Presentation Award, $150, 2006 9. UBC, Department of Pathology, 1 st Place Oral Award, $300,06/05 10. iCAPTURE Centre, Rookie of the Year Award, $100, 2003 11. BC Transplant Society, 1st Place Oral Award, $500, 2003  188  

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