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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 INCARDIAC ALLOGRAFT VASCULOPATHYbyARWEN LEIGH HUNTERB.Sc., The University of Victoria, 2002A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Pathology)THE UNIVERSITY OF BRITISH COLUMBIA(Vancouver)March 2008© Arwen Leigh Hunter, 2008AbstractHeart transplantation is often the only therapeutic option for patients with end stageheart disease. Allograft organs are in short supply. Thus, preserving the life of a graftedorgan is extremely important. Cardiac allograft vasculopathy (CAV) is an expression ofchronic rejection that accounts for the greatest loss of graft function in transplanted hearts.Peri-transplant ischemia/reperfusion (I/R)-injury occurs during transplantation when bloodflow is stopped to remove the heart from the donor and then is reinstated upon implantationof the donor heart into the recipient. This oxidative injury contributes to vascular dysfunctionand CAV. In this dissertation, I hypothesize that prevention and/or reduction of UR duringtransplantation reduces post-transplant vascular dysfunction and CAV. In this regard, myselfand my colleagues, examined the roles of apoptosis repressor with caspase recruitmentdomain (ARC) and cytochrome p450 (CYP) 2C enzymes in UR-induced vasculardysfunction 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. ARCoverexpression did protect against oxidant-induced cell death in H9c2 rat embryonicmyoblasts. We observed that ARC-overexpression prevented H9c2 differentiation intomuscle cells. With our focus on vascular injury, we turned our attention to the CYP 2Cenzymes. Both endothelium-dependent and independent vascular function was impairedfollowing I/R. Pre-treatment with the CYP 2C inhibitor sulfaphenazole (SP) restoredendothelial sensitivity to acetylcholine, but did not restore sensitivity to endothelium-independent vasodilators. Rat heterotopic heart transplants were performed with rats beingiitreated with SP or vector control prior to surgery. Rats treated with SP showed significantlyreduced luminal narrowing and had decreased SMC proliferation, oxidant and interferon-ylevels. No differences were detected in immune infiltration or apoptosis. Complementarystudies in cultured vascular cells revealed that CYP 2C9 expression decreased viability andincreased 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 anovel role for ARC in differentiation. CYP 2C contributes to post-ischemic vasculardysfunction and CAV through increased oxidative stress and endothelial dysfunction.iiiTable of ContentsAbstract^ iiTable of Contents ^ ivList of Tables viiList of Figures viiiList of Symbols, Abbreviations and Acronyms ^Acknowledgements ^ xiiCo-Authorship Statement xivChapter 1: Introduction 11.1 Cardiac Transplantation ^ 11.1.1 Historical perspective  11.1.2 Hyperacute and acute forms of cardiac transplant rejection ^ 51.1.3 Chronic cardiac transplant rejection by cardiac allograft vasculopathy (CAV) ^ 91.1.4 Vasomotor function following transplantation ^  161.1.5 Animal models of CAV ^ 181.2 Ischemia and Reperfusion (I/R) Injury ^ 201.2.1 Reactive oxygen and nitrogen species in I/R ^ 211.2.2 I/R and transplantation ^ 221.3 Apoptosis Repressor with Caspase Recruitment Domain (ARC)^241.3.1 ARC in I/R injury 251.4 Cytochrome p450 Enzymes (CYPs ^ 261.4.1 CYP 2C enzymes ^ 271.5 Arachidonic Acid (AA) Metabolism 361.5.1 AA metabolism by cyclooxygenase ^ 381.5.2 AA metabolism by lipoxygenase 381.5.3 AA metabolism by CYPs ^ 391.5.4 AA metabolites and cardiovascular disease ^ 401.6 Thesis objectives and hypotheses 411.7 Bibliography ^ 43Chapter 2: Apoptosis Repressor with Caspase Recruitment Domain in Vascular CellDeath and Myogenic Differentiation ) ^ 582.1 Introduction ^ 582.2 Aim 592.3 Materials and Methods ^ 602.3.1 Cell culture 602.3.2 RNA extraction and reverse transcriptase (RT)-PCR ^ 612.3.3 Cell lysis and Western blotting ^ 622.3.4 TAT protein expression, purification and Texas red staining ^ 632.3.5 TAT-fusion protein transduction and detection ^ 652.3.6 Cell viability^ 662.3.7 H9c2 stable and transient transfection ^ 672.3.8 H9c2 myocyte differentiation ^ 68iv2.3.9 F-actin and nuclear staining of H9c2 cells ^ 682.3.10 DEVDase activity assay ^ 692.3.11 Statistical analysis 692.4 Results ^ 702.4.1 Native ARC expression in endothelial and smooth muscle cell lines ^ 702.4.2 TAT-ARC purification and transduction in vascular cells ^ 702.4.3 ARC over-expression does not protect against H202 treatment. ^ 732.4.4 Functional overexpression of ARC in pre-differentiated H9c2 cells^ 732.4.5 Characterization of H9c2-ARC cell differentiation 772.4.7 Caspase-3 activation during H9c2 differentiation ^ 822.5 Discussion ^ 872.6 Bibliography 92Chapter 3: Cytochrome p450 2C Contributes to Post-Ischemic Vascular Dysfunction 2943.1 Introduction 943.2 Aim ^ 953.3 Materials and Methods ^ 963.3.1 Heart perfusion and vessel cannulation ^ 963.3.2 Vasomotor responses in septal arteries 973.3.3 Dihydroethidium (DHE) staining of coronary blood vessels ^ 973.3.4 Measurements of dityrosine in coronary effluents ^ 983.3.5 Statistical analysis ^ 983.4 Results ^ 983.4.1 Endothelium-dependent vasomotor responses 983.4.2 Endothelium-independent vasomotor responses ^  1003.4.3 Post-ischemic ROS production ^  1003.5 Discussion ^ 1073.6 Bibliography 111Chapter 4: Cytochrome p450 2C Contributes to Cardiac Allograft Vasculopathy3 .... 1134.1 Introduction 1134.2 Aim ^ 1154.3 Materials and Methods ^ 1154.3.1 Heterotopic heart transplantation ^  1154.3.2 Tissue collection  1164.3.3 Histological staining and immunohistochemistry (IHC) ^ 1174.3.4 Histological assessment and quantification ^  1184.3.5 Luminex analysis ^  1184.3.6 8-Isoprostane measurements ^  1194.3.7 Statistical analysis  1214.4 Results^ 1214.4.1 Post-surgical morbidity and mortality ^  1214.4.2 Assessment of CYP 2C6 expression in rat heart cross-sections ^ 1224.4.3 CYP 2C contributes to luminal narrowing in rat heterotopic heart transplants 1224.4.4 Assessment of general immune infiltration  1224.4.6 CYP 2C does not significantly alter post-transplant apoptosis ^ 1294.4.7 CYP 2C contributes to SMC proliferation following transplantation ^ 1374.4.8 Peripheral cytokine and chemokine levels following transplantation ^ 1374.4.9 CYP 2C contributes to serum 8-isoprostane levels ^  1424.5 Discussion ^ 1424.6 Bibliography 145Chapter 5: Cytochrome p450 2C9 in Vascular Cell Death and Oxidative Stress4 ^ 1485.1 Introduction ^ 1485.2 Aim 1495.3 Materials and Methods ^ 1505.3.1 Cell culture  1505.3.2 Cell lysis and Western blotting for CYP 2C9 ^ 1505.3.3 Adenoviral expression of CYP 2C9 in HUVEC 1515.3.4 Optimization of hypoxic conditions ^  1515.3.5 Cell viability in response to H/R 1525.3.6 Measurements of 8-isoprostane  1525.4 Results ^ 1535.4.1 Native, adenoviral, and H/R-induced expression of CYP 2C9 in HUVECs.... 1535.4.2 CYP 2C9 expression contributes to post H/R cell death in HUVEC ^ 1555.4.3 SP treatment does not alter SMC viability following H/R. ^ 1585.4.4 Effect of SP and COX-inhibition on 8-isoprostane production following H/R inCYP 2C9 expressing HUVECs ^ 1585.5 Discussion ^ 1615.6 Bibliography 164Chapter 6: Summary and Conclusions ^ 1666.1 Restatement of the Problem 1666.2 Summary of Findings ^ 1676.3 Relevance of Findings 1706.4 Future Directions 1716.5 Concluding Remarks ^ 1726.6 Bibliography 174Appendix I: Animal Care Certificate for Transplantation ^ 176Appendix II: Rat Heterotopic Heart Transplantation SOP 178Appendix III: List of Publications, Abstract, Oral Presentation and Awards ^ 186viList of TablesTable 1.1 2004 Revised ISHLT heart biopsy grading categories for cellular and antibody-mediated rejection. ^ 7Table 4.1 Peripheral cytokine and chemokine levels following heterotopic hearttransplantation in SP treated and control rats. ^  139Table 4.2 Repeated measures analysis of peripheral cytokine and chemokine levels followingheterotopic heart transplantation in SP treated and control rats. ^ 140viiList of FiguresFigure 1.1 ISHLT Kaplan-Meier survival curves for adult heart transplantation by era ^ 4Figure 1.2 Histology of CAV ^ 11Figure 1.3 Pathogenesis of CAV. 12Figure 1.4 The CYP monooxygenase reaction cycle ^ 28Figure 1.5 Overview of the three pathways of arachidonic acid metabolism^ 37Figure 2.1 A Map of the pTAT-HA-fusion protein. 64Figure 2.2 HCASMCs and HUVECs express ARC. 71Figure 2.3 TAT-ARC fusion protein transduction into HCASMCs and HUVECs. ^ 72Figure 2.4 TAT-ARC uptake into HUVECs and HCASMCs is punctate and concentration-dependent. ^ 74Figure 2.5 TAT-ARC does not confer greater protection against H202 in HUVECs than TAT-0-gal control 75Figure 2.6 TAT-ARC does not confer greater protection against H202 in HCASMCs thanTAT-0-gal control ^ 76Figure 2.7 Overexpression of ARC prevents H202-induced cell death. ^ 78Figure 2.8 Overexpression of ARC prevents myogenic differentiation. 79Figure 2.9 Overexpression of ARC prevents myogenic differentiation. 80Figure 2.10 Overexpression of ARC prevents myogenic differentiation. ^ 81Figure 2.11 ARC stable overexpression prevents the expression of the muscle-specificmarkers troponin T and myogenin. ^ 83Figure 2.12 Transient ARC overexpression prevents the expression of the muscle-specificmarkers troponin T and myogenin. 84Figure 2.13 ARC levels increase in H9c2 cells upon differentiation. ^ 85Figure 2.14 ARC overexpression prevents caspase-3/7 activity during differentiation. ^ 86Figure 3.1 Sulfaphenazole (SP) restores post-ischemic endothelium-dependent NO-mediatedvasodilation. ^ 99Figure 3.2 SP does not restore post-ischemic endothelium-independent vasodilation producedby sodium nitroprusside (SNP). ^  101Figure 3.3 SP does not restore post-ischemic endothelium-independent vasodilation producedby isoproterenol. ^  102Figure 3.4 Constrictor responses to KCl were unaffected by SP pre-treatment. ^ 103Figure 3.5 SP reduces ROS production following UR ^ 104Figure 3.6 SP reduces ROS production following UR 105Figure 3.7 Peroxynitrite measurements in post-ischemic coronary effluent. ^ 106Figure 3.8 Proposed mechanism of CYP 2C induced impaired post-ischemic vasodilation. 109Figure 4.1 SP treatment does not reduce post-transplant weight gain.  123Figure 4.2 CYP 2C6 is expressed in transplanted rat heart blood vessels and myocardium.124Figure 4.3 SP administration at time of surgery attenuates allograft luminal narrowing ^ 125Figure 4.4 SP administration at time of surgery attenuates allograft luminal narrowing ^ 126Figure 4.5 Histological features of diffuse and focal myocardial infiltration ^ 127Figure 4.6 Histological features of epicardial and endocardial immune infiltration. ^ 128Figure 4.7 CYP 2C does not contribute to general myocardial immune infiltration. ^ 130Figure 4.8 CYP 2C does not contribute to perivascular immune infiltration. ^ 131viiiFigure 4.9 Perivascular immune infiltration in the absence of luminal narrowing. ^ 132Figure 4.10 SP treatment does not alter CD3 + lymphocyte infiltration in the vasculature ^ 133Figure 4.11 SP treatment does not alter CD8 + lymphocyte infiltration in the vasculature ^ 134Figure 4.12 CYP 2C does not significantly contribute to post-transplant apoptosis. ^ 135Figure 4.13 CYP 2C-inhibition has an insignificant effect on EC loss at day 4 post-transplant.^  136Figure 4.13 CYP 2C contributes to SMC proliferation. ^  138Figure 4.14 CYP 2C contributes to peripheral IFN-y levels post-transplantation ^ 141Figure 4.15 CYP 2C contributes to post-transplant serum free 8-isoprostane levels. ^ 143Figure 5.1 Relationship between hypoxic chamber oxygen concentration and measured P02.^  154Figure 5.2 CYP 2C9 expression in HUVECs following adenoviral transfection and H/R ^ 156Figure 5.3 CYP 2C9 expression in HUVECs reduces cell viability following H/R ^ 157Figure 5.4 SP treatment in HCASMCs does not alter proliferation or cell viability followingH/R^ 159Figure 5.5 CYP 2C increases 8-isoprostane levels. ^  160ixList of Symbols, Abbreviations and AcronymsA^AA^Arachidonic acidACh AcetylcholineAIDS^Acquired immune deficiency syndromeAM AcetoxymethylAMR^Antibody-mediated rejectionARC^Apoptosis repressor with caspase recruitment domainC^[Ca2-1c^Intracellular calcium levels[Ca21„,^Mitochondrial calcium levelsCARD^Caspase recruitment domainCAV^Cardiac allograft vasculopathyCK Creatine kinaseCMV^CytomegalovirusCOX^CyclooxygenaseCYP Cytochrome p450D DC^Dendritic cellDEA^Dihydroxyeicosatraenoic acidDHE^DihydroethidiumDMEM^Dulbecco's modified eagle's mediumE E(B/G)M Endothelial basal/growth mediumEC^Endothelial cellEDHF^Endothelium derived hyperpolarizing factorEET Epoxyecosotrienoic acidEGF^Endothelial growth factoreNOS^Endothelial nitric oxide synthaseERK^Extracellular signal-regulated kinasesF^F344^Fisher 344 ratFBS Foetal bovine serumFCS^Foetal calf serumG GA-1000 Gentomycin-amphotericin BGM-CSF^Granulocyte macrophage colony-stimulating factorGRO/KC Growth-related oncogeneH H/R^Hypoxia and re-oxygenationH2O2^Hydrogen peroxideHAR Hyperacute rejectionHCASMC Human coronary artery smooth muscle cellHE 1E^Hydroxyecosatraenoic acidHLM^Human liver microsomesHPE1E^Hydroperoxyeicosatraenoic acidHS^Horse serumHUVEC^Human umbilical venous endothelial cellI/R^Ischemia and reperfusionICAMIFN-yIHCILiNOSiPLA2ISHLTIVUSL LOXLTM^MAPKMCPMHCMIMTSN NeoNF-KBNKNO O 02- ON00-OxLDLP PARPPECAMPGPGI2PKCPLA2PVSR RNSROSRT-PCRS^SERCASm(B/G)MSMCSNPSNPSODSPT^TATTNF-aTXIntracellular adhesion markerInterferon gammaImmunohistochemistryInterleukinInducible nitric oxide synthaseInducible phospholipase A2International society for heart and lung transplantationIntravascular ultrasoundLipoxygenaseLeukotrienesMitogen activated protein kinaseMonocyte chemoattractant proteinMajor histocompatibility complexMyocardial infarctionCellTiter96 AQuoeus assayNeomycinNuclear factor kappa BNatural killerNitric oxideSuperoxidePeroxynitriteOxidized low density lipoproteinPoly (ADP-ribose) polymerasePlatelet endothelial cell adhesion moleculeProstaglandinProstacyclinProtein kinase CPhospholipase A2Perivascular spaceReactive nitrogen speciesReactive oxygen speciesReverse transcriptase - polymerase chain reactionSarco/endoplamsic reticulum calcium ATPaseSmooth muscle basal/growth mediumSmooth muscle cellSingle nucleotide polymorphismSodium nitroprussideSuperoxide dismutaseSulfaphenazoleTransactivator of transcriptionTumour necrosis factor alphaThromboxaneAcknowledgementsFirstly, 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 barhigher and push to obtain my goals. He has also challenged me to think and workindependently and to take the lead in my research. As a result, Dave has put me on my waytowards becoming an innovative and productive researcher. He has also encouraged me toalways keep my eyes open, never to discount unexpected findings and, of course, hasimparted his wisdom about the importance of a good lunch. I would also like to thank my co-supervisor Dr. Bruce McManus. Bruce's immense understanding of inflammatorycardiovascular diseases, including cardiac allograft vasculopathy, has been a tremendousasset 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 andStroke Foundation (H&SF) of BC and Yukon for providing operating funds supporting thisresearch. Thank-you to the H&SF of Canada, The Michael Smith Foundation for HealthResearch (MSFHR) and The Canadian Institutes for Health Research/MSFHRTransplantation 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 theTAT-ARC Construct and the CYP 2C9 adenoviral constructs, respectively. Thanks also go toAmrit Aitken for assistance with staining and sectioning and Dr. Ryon Bateman forassistance with P02 measurements. A tremendous thanks to all 'the Granvillites': HongyanxiiZhoa, Rani Cruz, Wendy Boivin, Ciara Chamberlain, Lisa Ang and Paul Hiebert; and to allour 'honorary Granvillites'; most notably, Erin Tranfield for their day to day help and formaking the laboratory a great place to be everyday. I owe a debt of gratitude to the co-operative education and summer students that I have worked with throughout my studies:Shirley Chen, Kellyann Jones, Eric Venos, Munreet Chehal, Katelyn Mueller and PaulHiebert. I also owe a special gratitude to the late Dr. Sasha Kerjner whose surgical expertisein heterotopic transplantation was invaluable to this project and whose sense of humour andkindness will ensure her a warm place in my memories.Finally, I would like to thank my family. Thank-you to my parents, Ann and DonHunter, my sisters, Carly and Sarah, and their husbands, Jon and Beau, and Damon for theirlove, support and encouragement.Co-Authorship StatementChapter 2 is based on the manuscript "Apoptosis repressor with caspase recruitment domain(ARC) inhibits myogenic differentiation." It was published in FEBS Letters, 2007, volume581(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. DavidJ. Granville. I worked with the senior authors on this paper to develop the research plan forthis paper. I was assisted by Jingchun Zhang and Dr. Xiaoning Si with the adenoviralinfection experiments and quantitation of multinucleation. Shirley Chen was a co-operativeeducation student under my supervision and assisted with collection of cell lysates andWestern blotting analyses on experiments related to stably transfected cells. Brian Wongprovided valuable assistance in experiments related to immunofluorescence staining. Dr.Daryoush Ekhterae provided the stably transfected cell lines for this paper. Drs. Luo andGranville are co-senior authors on this publication. I was primarily responsible for writingthe 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 responsiblefor heart isolation, isolation of septal arteries and artery cannulation. Drs. Laher andGranville 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 writingxivof the manuscript. I performed all assays assessing reactive oxygen species production andassisted with assays related to induction of ischemia and reperfusion and measurement ofvascular 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 AmericanJournal of Transplantation. This manuscript was co-authored with Dr. Alexandra Kerjner,Katelyn Mueller, Dr. Bruce McManus and Dr. David Granville. Dr. Kerjner performed thesurgical aspects of the rat heterotopic cardiac transplantation. Katelyn Mueller was a co-operative student under my supervision who assisted with some of the immunohistochemicalstudies. Dr. McManus provided insight into the grading of the immune infiltration. Dr.Granville is the senior author on this publication. I developed the experimental approachtogether with the senior author and helped to write the grant that funded this research. Iconducted the bench work and wrote the paper which was reviewed and edited by the seniorauthor.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 undermy supervision that assisted with measurements of arachidonic acid metabolites. Dr.Granville is the senior author on this publication. I, along with the senior author, developedthe research plan for this project and carried out the experimental protocols described in thischapter.xvChapter 1Chapter 1: Introduction1.1 Cardiac Transplantation1.1.1 Historical perspectiveAlthough organ transplantation became a viable therapeutic strategy only in the pasttwenty-five years, the concept of exchanging organs and tissues between individuals hasexisted for millennia. Early references to organ transplantation include the Chinese physicianPien Ch'iao reportedly exchanged hearts between a man of weak will and a man of strongwill in 500 B.C. and in the third century A.D. the Roman Catholic saints Damian and Cosmasreportedly replaced the gangrenous leg of a Roman Deacon with the leg of a recentlydeceased 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 majoradvancements in the field of transplantation occurred. This section does not aim to provide acomplete history of transplantation but simply to highlight major findings that furthered theadvancement of the field and underline those areas for which significant research is needed.The first systemic study of transplantation occurred in 1908 when Alexis Carrelperformed double kidney exchanges between cats. This study was made possible due to thedevelopment, by Carrel with Charles Guthrie, of the technique of artery and veinanastomoses. 2 This technique, still used today, laid the groundwork for solid organtransplantation and many other vascular procedures and won Alexis Carrel the Nobel Prize inPhysiology or Medicine in 1912. Although none of the cats in Carrel's study survived, some1Chapter 1were able to maintain urinary output for up to 25 days, thus demonstrating that organtransplantation was viable at the surgical level. The discovery of human ABO blood groupsby Karl Landsteiner in 1900 combined with the hypothesis of Peter Medawar that transplantrejection was an immunological process3 lead to the first successful solid organ transplant;performed in 1954 by Joseph Murray, involving a kidney transplant between identical twins. 4Unlike other solid organ transplants, cardiac orthotopic transplantation was notsurgically 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 transplantin 1964. Unfortunately the recipient's heart failed prior to a human donor becoming availableso Hardy proceeded using a chimpanzee heart which quickly failed due to hyperacuterejection (described in section 1.2.1). 5 In 1967, Christiaan Barnard performed the firstsuccessful heart transplant in South Africa with the recipient surviving for eighteen daysfollowing transplantation before dying as a result of pneumonia. 6 This lead to over 100 hearttransplants being performed in the late 1960s. 7 Unfortunately the results were disappointingwith a mean survival of only 29 days and many centres discontinued their cardiactransplantation programs. 3 However, during this time Dr. Norman Shumway, at StanfordUniversity, continued programs in both transplantation research and clinical transplantation.His team developed techniques in simplified orthotopic surgical procedures, organpreservation by hyperthermia and rejection monitoring by electrocardiography and serialbiopsy.3 By the end of the 1970s the Stanford transplantation program had improved their 1year survival level from 22% to 65%. 8 For his efforts, Dr. Shumway is widely considered thefather of modern clinical cardiac transplantation.32Chapter 1The discovery of potent immunosuppressive drugs was equally as important in thehistory of transplantation as the aforementioned surgical advances. As early as 1951, PeterMedawar, working for the National Institute for Medical Research, suggestedimmunosuppressive drugs could be used in transplantation.9 However, the drugs available atthe time; namely, cortisone and azathioprine, were not strong enough immunosuppressors formost types of transplantation. In 1980, a sufficiently potent immunosuppressor wasdiscovered in cyclosporin. 1015 Since that time, many other important immunosuppressivedrugs 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 thesociety annually. 16 Of those transplants reported, heart transplant recipients can now expect a1-year survival rate approaching 90% and an average graft life of 10.3 years (Figure 1.1). 16Despite these impressive achievements, the field of cardiac transplantation still has a longway to go. Malignancies and infections due to immunosuppressive regimes is currently thelargest cause of death amongst transplant recipients and the largest impediments to long termgraft survival is chronic heart transplant rejection in the form of cardiac allograftvasculopathy (CAV, described in detail in section 1.1.3). 17 The pathogenesis of CAV is thecentral focus of this thesis.3Chapter 1100All comparisons significant at p < 0.000180^-•60CO40 — -CO20HALF-LIFE 1982 -1991: 8.9 years; 1992-2001: 10.3 years; 2002-6/2005: NA00^1^2 3^4 5^6 7^8 9 10 11 12 13 14 15- - - 1982-1991 (N=18,844)— - 1992-2001 (N=34,987)2002-6/2005 (N=9,459)••YearsFigure 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 asillustrated by the ISHLT registry slides. 17 Graph incorporates information from all transplantswhere 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.4Chapter 11.1.2 Hyperacute and acute forms of cardiac transplant rejectionTransplant rejection is the process by which the transplant recipient's immune systemrecognizes 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, acuteantibody-mediated rejection and CAV. This section aims to provide a brief overview of thehyperacute and acute forms of allograft rejection. As CAV is central to this thesis, a moredetailed discussion of its pathological features and aetiology is provided in section 1.1.3.1.1.2.1 Hyperacute rejectionHyperacute rejection (HAR), also termed antibody-mediated rejection, describes theprocess of graft destruction within minutes to hours following transplantation. 18 HAR iscaused when the recipient has pre-formed circulating antibodies to endothelial antigenspresent on the graft; most commonly ABO blood group antigens or major histocompatibilitycomplex (MHC) antigens. 19 The formation of antigen-antibody complexes activates thecomplement system causing mass neutrophil infiltration, endothelial damage and subsequentmicro-thrombi. 19 HAR is now rarely observed in allografts (i.e., grafts between two membersof the same species) but can occur if recipients have previously been exposed to MHC-antigens present on the graft from prior pregnancies, blood transfusions or transplants orwhen errors in ABO blood type matching occur. An interesting exception to this happens ininfants where ABO blood type matching may not be required. Infants do not produceisohemagglutinins or serum anti-A or anti-B antibodies until 12-14 months of age. 20 Hearttransplants have been successfully performed in infants across the ABO barrier without5Chapter 1incident of HAR. 21-23 Unlike with allotransplantation, HAR remains a major hurdle inxenotranplantation (i.e. transplantation between two members of different species) andremains an area of intense research in this field.1.1.2.2 Acute cellular rejectionAcute cellular rejection is a process that can occur starting only days followingtransplantation but can also occur at any time during the life of the graft. Between 40 and50% of all transplant recipients will be treated for at least one acute rejection episode withina year of receiving their transplants. 17 Although increasing levels of immunosuppressivedrugs are able to combat the majority of acute rejection episodes, over-immunosuppressingpatients can lead to malignancies and infections and therefore must be kept in balance.Acute cellular rejection requires alloreactive T-lymphocytes (either CD4 + or CD8 ±) torecognize alloantigens expressed on the graft resulting in their activation and subsequentproliferation. Immune infiltration then leads to graft cell necrosis and or vessel thrombosisand eventually graft function loss. 24 The endomyocardial biopsy was first described by Caveset al. in 1973 as a method of monitoring cellular transplant rejection. 25 It remains the 'goldstandard' for monitoring cardiac transplants for signs of rejection. The ISHLT first created agrading scale for histological diagnosis of acute rejection in 1990. 26 This grading scaleremained unchanged until it was updated by the Society in 2004. 27 The criteria for theupdated grading scale are shown in Table 1.1.Immunosuppressive strategies for rejection vary per transplant centre and oftenamong patients within each centre. Most commonly, triple-drug therapy and cytolytic6Chapter 1Grade code^ Grade criteriaCellular rejectionGrade 0 R No rejectionGrade 1 R, mild Interstitial and/or perivascular infiltrate with up to 1 focus ofmyocyte damageGrade 2 R, moderate Two or more foci of infiltrate with associated m oc to damageGrade 3 R, severe Diffuse infiltrate with multifocal myocyte damage ± edema, ±hemorrha!e ± vasculitisAntibody-Mediated Rejection (AMR)AMR 0 Negative for acute AMRNo histological or immunopathological features of AMRAMR 1 Positive for AMRHistological feature of AMRPositive immunofluorescence or immunoperoxidase staining forAMR (positive CD68, C4d)Table 1.1 2004 Revised ISHLT heart biopsy grading categories for cellular andantibody-mediated rejection.Standardized cardiac biopsy grading for acute cellular rejection and acute antibody-mediatedrejection as modified from Stewart et al. 27 'R' denotes revised grade to differentiate thesegrades from the 1990 criteria. 267Chapter 1therapy are utilized during the peri-operative period. Triple-drug therapy includes preventionof lymphocyte differentiation by interleukin-2 (IL-2) reduction through cyclosporine ortacrolimus, purine synthesis inhibition by azathioprine or mycophenolate mofetil, andlympholytic treatment with corticosteroid therapy. Cytolytic agents employed include OKT3and ATG/ALG. To reduce the negative impact of the various side effects of each of thesetreatments doses are decreased following the peri-operative period constituting maintenanceimmunosuppression. (Reviewed in 28)1.1.2.3 Antibody-mediated rejectionAntibody-mediated rejection (AMR), also termed 'biopsy-negative rejection',`vascular rejection' and 'humoral rejection', is a form of vascular inflammation or damageresulting in hemodynamic compromise where there is minimal evidence of cellularrejection.29-33 Surprisingly, patients in this category have worse outcomes than patients withhigher ISHLT biopsy scores. 34 AMR does not typically respond well to increasedimmunosuppressive therapy and increases risk of graft loss, CAV and mortality.AMR is characterized by prominent capillaries in the biopsy that have endothelialswelling and deposition of immunoglobulin and complement. 29 Endothelial swelling of thecapillaries results from macrophage influx into injured capillaries and thus can be detectedusing macrophage markers, most commonly CD68. 35' 36 AMR is typically diagnosed aspresent or absent based on the ISHLT guidelines shown in Table 1.1. Current treatmentregimes for AMR are limited and usually involve high-dose corticosteroids with more severecases also requiring cytolytic agents, such as OKT3, and thymoglobulin or gamma globulin,8Chapter 1heparin and antiproliferative agents. 37 Other immunosuppressive drugs including tacrolimus,mycophenolate mofetil and sirolimus have also been used with some success against AMR. 38'391.1.3 Chronic cardiac transplant rejection by cardiac allograft vasculopathy (CAV)CAV is the greatest cause of graft loss for heart transplant recipients surviving 1 yearfollowing transplantation. 17 Because transplanted hearts are largely denervated, transplantpatients do not experience typical sensations associated with myocardial ischemia orinfarction. Therefore, the first clinical indications of CAV can be arrhythmias, congestiveheart 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 Intravascularultrasound is able to detect much higher levels of CAV with transplant related intimalthickening being detected in 75% of transplant recipients only 1 year followingtransplantation.421.1.3.1 Pathological characteristics of CAVCAV is a form of arteriosclerosis characterized by diffuse and obliterative intimalthickening. Although CAV is classically described as concentric, fibrous plaques, there are awide array of abnormal phenotypes including lesions resembling complicated nativeatherosclerosis. 43 In the latter stages of development allograft arteriosclerosis often involveslipid deposition and calcification. 44' 45 Further complicating histological phenotyping is thatCAV can occur in regions with existing atheromatous disease. This phenotype is likely to9Chapter 1become more common with transplant programs accepting hearts from donors over 55 yearsof age, considered marginal donors. CAV involves large and small epicardial and intramuralarteries as well as venous structures of the graft. 46 '47 The recipients native blood vessels arenot 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 andmay experience thinning.48 Figure 1.2 shows the classical phenotype and some of thecommon pathological characteristics of CAV.1.1.3.2 Pathobiology of CAVThe pathogenesis of CAV, shown in Figure 1.3, is not yet fully elucidated but isbelieved to involve a chronic allogenic response to the transplanted organ propagated by non-immunological factors.49 The importance of the immune system to the development of CAVis evident by the absence of CAV development in isografts. 40 The endothelium of the graftedheart serves as an interface between the allograft and the recipient. Endothelial cells are thefirst cells to be recognized and are the primary target of the hosts immune system. 49 Non-immunological factors, as well as, prior episodes of acute and antibody-mediated rejectioncan contribute to endothelial activation early following transplantation. Although endothelialactivation remains a loosely defined term it generally involves increased expression and/orpresentation of MHC antigens, adhesion and co-stimulatory molecules, along with alteredsecretion of cytokines and chemokines. 5° Endothelial activation enhances entry of immunecells, decreases endothelial adhesiveness, contributes to impaired vasomotor function,described in section 1.1.4, and intimal thickening. 51 Both cell-mediated and10Chapter 1Figure 1.2 Histology of CAV.Distal left anterior descending artery from a 16 year old male, 20 months post-transplantfrom a 47 year old male donor. The artery shows a narrowed lumen (L), a recent, nearlyocclusive 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) andproteoglycan (PG) deposits. The adventitia (A) has multiple enlarged vaso vasora (examplesshown with arrows).1 IPropagating endothelial InjuryI/R injuryMetabolic abnormalitiesCMV infectionAntibody-mediated rejectionCellular rejection damageNon-denuding injuryDenuding injuryLumenEndotheliumlntimaLuminal narrowing_owI^MHC & EAexpressionNOSMC and myofibroblastmigration and proliferationElastic lamenaLipidsChapter 1Figure 1.3 Pathogenesis of CAV.Upon propagating endothelial injury induces both denuding and non-denuding injury of theendothelium. Denuding injury increases endothelial permeability allowing increased entry ofT-lymphocytes (T), monocytes and macrophages (M) and lipids further contributing tovascular damage. Non-denuding injury induces MHC expression and presentation ofendothelial antigens (EA). Immunogenic endothelium allows antigen expression by dendriticcells (D) and consequent activation and differentiation of T-cells. Non-denuding injury alsoleads to decreased nitric oxide (NO ) production increasing SMC and myofibroblastmigration and proliferation in the neo-intima.12Chapter 1humoral responses have been found to play roles in CAV.Cell-mediated responses primarily involve recognition of MHC antigens throughantigen 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 donorendothelial cells.52 These professional antigen presenting cells can then activate largenumbers of T-lymphocytes. Endothelial cells constitutively express both class I and class IIMHC antigens and are therefore able to activate CD8 + and CD4+ lymphocytes, respectively,through indirect allorecognition. The degree of MHC antigen mismatch has been found tocorrelate with the development of CAV. 53' 54 During allograft rejection MHC class II isupregulated further enhancing recognition by CD4 + lymphocytes. 55 CD4+ lymphocytes uponactivation release IL-2 stimulating the generation of CD8 + cytotoxic lymphocytes, activatealloantibody-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 themechanisms of CD4 ± induced allograft intimal thickening have indicated that a Thl-typeresponse contribute to, 57' 58 and a Th2-type response may protect against59 CAV.Although MHC molecules are important contributors to CAV development, they arenot 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 developCAV.61 Antibodies against multiple and diverse endothelial antigens have been detected intransplant recipients and have been linked to graft vasculopathy. 62-64 Vimentin has beenfound as the most prominent antigen in CAV and patients with high levels of anti-vimentinantibodies are at increased risk of graft arteriopathy. 64-66 Antibodies against intracellular13Chapter 1adhesion marker (ICAM)-1 has also been linked to CAV and polymorphisms in ICAM-1may be protective against rejection. 67Upon 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 myofibroblast68proliferation, fibrosis and infiltration of macrophage/monocytes and T-lymphocytes. 4° Thesecells are also able to alter cytokine and chemokine levels and augment extracellular matrixsynthesis. 24 Endothelial cells in healthy vessels produce nitric oxide (NO ) and anti-thrombotic proteins. 69 NO  plays a vital role in vascular homeostasis, described in section1.1.4, 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 theNO  synthase pathway leading to impaired NO  production and activity. 51 Endothelial NO synthase (eNOS) deficient mice have accelerated allograft vasculopathy in aortictransplants.74Several cytokines and chemokines are involved in the pathogenesis of CAV.Interferon-gamma (IFN-y) is thought to be a key regulator of graft arteriosclerosis. (Reviewed in75) IFN-y is primarily produced by Thl CD4+ T-cells following induction by IL-12 but canalso be produced by CD8 + T-cells, natural killer (NK) cells, NK T-cells, DC andmacrophages. 75 As mentioned above, transplants in mice with a deficient Thl-type responseand in IFN-y knockout mice have reduced CAV.57' 58 IFN-y regulates hundreds of genesincluding pro-inflammatory cytokines and chemokines, growth factors, transcription factorsand membrane receptors. 76 IFN-y enhances expression of MHC class I and II molecules 77' 78and induces leukocyte independent SMC proliferation in vascular transplants. 79 Thl CD4+ T-cells also produce IL-2 involved in T-cell expansion. RAN 1ES is an IFN-y-induced14Chapter 1chemokine which increases monocyte adhesion of activated graft endolium. 8° Monocytechemoattractant protein (MCP)-1, MCP-3 and IL-8 have also been detected in CAVlesions.5° IL-10 and other Th2-type response cytokines, including IL-4, 5, 6, 9, and 13 appearto be protective against CAV development. 59Non-immunological factors known to contribute to CAV development includehyperlipidemia, viral infection, immunosuppressive drug toxicity and ischemia andreperfusion (I/R) injury. Hyperlipidemia promotes development of fibrotic intimalhyperplasia and lipid deposition in the native vasculature and, at an accelerated rate, in thevasculature of the allograft. 81 Prospective assessment of simvastatin therapy in transplantrecipients has demonstrated that statins increase the survival of transplant patients anddecrease the development of CAV by more than 45% 11 years post-transplant. 82Cytomegalovirus (CMV) is the most common viral infection amongst transplant patients. 5°CMV infection has been linked with CAV development though activation of NF- KB andsubsequent production of pro-inflammatory cytokines and SMC proliferation. 83-86 Anti-CMVtherapy with CMV hyperimmune globulin and ganciclovir reduced both CMV titres andcoronary artery luminal narrowing in heart and heart-lung transplant recipients. 87 Mostimmunosuppressive drugs have little protective benefit against CAV and their side effectssuch as hyperlipidemia, glucose intolerance and hypertension may actually contribute to itsdevelopment. 88 I/R injury and its relationship to CAV development are discussed in detail insection 1.2 and 1.2.2, respectively.15Chapter 11.1.3.3 Treatment of CAVTreatments to control risk factors, such as CMV infection and hyperlipidemia, mayhelp prevent CAV. Unfortunately, there are relatively few treatments for established CAV. Inpatients where the CAV is focal and has not spread throughout the vascular tree,percutaneous and surgical interventions may be useful. Percutaneous transluminal coronaryangioplasty and coronary artery stenting are used by some transplant programs to extendgraft 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 transplantpatients that received stents. 92 Coronary artery bypass grafting in patients with CAV carriesabnormally high risk of perioperative death with death rates between 33.3% and 40% atvarious transplant centers. 93-95 For patients with diffuse CAV, retransplantation is often theonly option. Among immunosuppressive drugs, sirolimus appears to offer the mostprotection against CAV development and progression by reducing smooth muscleproliferation and migration, increased NO  production, decreased angiogenesis, andinhibition of fibrosis and extracellular matrix production. 96 Ultimately, preventing the factorsthat propagate CAV development would offer the greatest advantage to transplant recipients.1.1.4 Vasomotor function following transplantationPost-transplant abnormalities in endothelium-dependent vasomotion can be detectedearly following transplantation in both macro and micro vessels. 51 Some of theseabnormalities can be indicative of peri-transplant endothelial denudation or dysfunction.Transplanted organs often have partial vasoconstrictory, rather than a vasodilatory, responses16Chapter 1to acetylcholine. 97. 98 This phenomenon may resolve in the months followingtransplantation. 99 However many groups have detected impairments in vasodilatoryresponses to acetylcholine, substance P, exercise, serotonin, and cold-pressor testing bothimmediately following transplantation and years post-transplant. 100-102 Reduced eNOSexpression post-transplant is reported to contribute to impaired acetylcholine-inducedvasodilation. 1°3 Endothelium-independent vasodilation is also impaired following hearttransplantation. This impairment may be caused by cytotoxic damage to the vascular SMCsleading to medial thinning 104 impairedmpaired SMC contractile responses l°5.1.1.4.1 Early vasomotor dysfunction as a predictor of CAVAlthough some researchers have not found a correlation between early vasculardysfunction and CAV development99, other studies have found that early vasculardysfunction is predictive of CAV. Davis et al. 106 found that quantitative angiographymeasurements of acetylcholine-induced vasodilation correlated with CAV development 1-year following transplantation as detected by intravascular ultrasound (NUS). Hollenberg etal. 107 also found a correlation between impaired acetylcholine responses and CAVdevelopment detected using angiography. In the latter study, responses to adenosine andnitroglycerine were also assessed but were not found to correlated with future CAVdiagnoses. 1°717Chapter 11.1.5 Animal models of CAVAnimal models for CAV include orthotopic arterial grafting and heterotopic andorthotopic cardiac transplantation. All of these models expose the grafted tissue to therecipient's immune system and have provided valuable insight into CAV's aetiology. Theadvantages and disadvantages of these models are briefly described below.Orthotopic arterial grafting involves harvesting a section of a major artery, typicallythe aorta or carotid artery, from a donor and inserting the artery into the identical position inthe recipient's arterial system using end-to-end anastomoses. 108 This model has manyadvantages, not the least of which is that it is surgically less challenging and can be used insmall animal models with lower post-surgical morbidity or mortality (< 2%) 108 than cardiactransplantation. Arterial grafts develop classical features of CAV including intimalthickening, smooth muscle proliferation and immune infiltration. 108 These arteries aremaintained at near physiological conditions, unlike with heterotopic transplantation; however,they lack the parenchymal factors that can contribute to rejection. This technique precludesmeasurements of organ function and associative studies of acute rejection and CAVdevelopment.Heterotopic cardiac transplantation involves grafting of a donor heart into a non-physiological position, most commonly the abdominal cavity, of the recipient. A detailedsurgical protocol for abdominal rat heterotopic cardiac transplantation is provided inAppendix I. Similarly to the orthotopic arterial graft model, heterotopic cardiac grafts can beperformed in small animal models. This allows researchers to study CAV in inbred andgenetically altered populations. This model is advantageous over arterial grafting as it allows18Chapter 1evaluation of acute rejection and graft survival can be monitored easily and non-invasivelyby abdominal palpation. 1°9 The disadvantage of heterotopic heart transplantation is that thehearts are not physiologically loaded and perfusion of the myocardium by the recipient'scirculatory system is retrograde through the ostia and into the coronary circulation.Orthotopic cardiac transplantation parallels human orthotopic heart transplantationand offers the advantages that the hearts are physiologically loaded and that parenchymaleffects and acute rejection can also be examined. Unfortunately, the surgical aspects of thisprocedure are inhibitory in most cases. Specifically, a heart-lung bypass machine is requiredand thus this operation is generally restricted to canine, swine and other large mammalmodels. The expense of using large animal models and the lack of genetic and inbredpopulations makes use of this model rare.1.1.5.1 Lewis to Fisher 344 rat model of heterotopic heart transplantationWe selected the Lewis to Fisher 344 (F344) rat heterotopic heart transplant model forthese studies. As mentioned in section 1.1.3.2, Lewis and F344 rats have identical class I andII 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-F344transplant model was originally described as a CAV model in 1993 by Adams et al. 112 Thismodel was ideal for this study as it is a well-established model allowing for comparisonswith previous studies. It also utilizes commercially available rat strains, has long-survivinggrafts 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 CAV19Chapter 1lesions consisting largely of intimal SMC accumulation. A slight increase in monocellularinfiltration and necrosis are observed compared to human CAV. 1121.2 Ischemia and Reperfusion (UR) InjuryI/R is integral in the pathophysiology of myocardial infarction and is a contributingcomplication to multiple surgical procedures including cardiac transplantation and coronarybypass. 113-118 I/R results in apoptosis and necrosis in the myocardium. The vascularendothelium is even more susceptible to ischemic damage. 119 I/R is associated withdecreased endothelium-dependent vasodilation, decreased NO levels, increased expressionof MHC, adhesion molecules and leukocyte adhesion 120, and adverse contractile modulatoryeffects. 121Ischemia is insufficient or absent blood flow and reperfusion is restoration of bloodflow flowing an ischemic period. During ischemia, ATP supplies are depleted leading to anincrease in cytosolic calcium ([Ca2+]c). Elevated [Ca2+], can be prolonged due to reperfusioninjury 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 [Ca21c inturn causes an increase in mitochondria' calcium ([Ca 2±],n) levels associated with increasedproduction of ROS linked to damage of the respiratory chain. 12320Chapter 11.2.1 Reactive oxygen and nitrogen species in URROS and RNS are central players in the pathogenesis of UR. ROS and RNS can bebroadly divided into free radicals (1-electron donors) and non-radical oxidants (2-electrondonors). Free radical oxidants, such as superoxide (02-) and NOV,  can be highly reactive andcan act as both oxidizing and reducing agents as they are capable of both donating andaccepting 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 throughinducible NO synthase (iNOS). As described above, NO plays important roles in vascularhomeostasis through its induction of endothelium dependent vasodilation, plateletaggregation, and in controlling smooth muscle growth and differentiation. However, whenNO- is produced in the presence of 02-  the two rapidly react in the formation of ONOO-, anon-radical oxidant. 124 Non-radical oxidants are capable of accepting two electrons. Otherexamples of such oxidants include: hydrogen peroxide (H202), ozone and hypochlorous acid.Non-radical oxidants, like ONOO-, are highly chemically reactive and known to cause celldamage through lipid peroxidation, tyrosine nitration, and reactions with sulfhydrylgroups. 125 In addition to its ability to chemically alter many cellular components, ONOO- canalso cause cellular dysfunction through the activation of multiple signalling pathways.ONOO- has been shown to increase integrin-dependent adhesion of human neutrophils tohuman coronary artery endothelial cells through activation of the Raf-1/ extracellular signal-regulated 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 activatedprotein kinase (MAPK). 126 ONOO- can also induce apoptosis. 127-133 Previous research by ourcentre has shown that acute cardiac rejection and apoptosis is attenuated when mouse cardiac21Chapter 1allografts are transplanted into iNOS knockout recipients compared to iNOS+/+ recipients. 134Furthermore, 02-  and ONOO- can disrupt ER calcium ATPases and Ca 2+ regulation incoronary arteries. 122' 135-141 ONOO- is also implicated in smooth muscle cell damage throughDNA damage and the activation of poly(ADP)ribose synthetase which results in energydepletion. 142ROS and RNS are generated through multiple pathways including: NAD(P)H oxidase,xanthine oxidase, myeloperoxidase, lipoxygenase, mitochondrial respiration, transitionmetals, and nitric oxide synthase (NOS). Although they are largely unstudied outside of thehepatic system, cytochrome p450 enzymes (CYPs) can also generate ROS and lead to theproduction of RNS in cardiovascular tissues. Production of ROS by CYPs is discussed insection 1.4. Antioxidants are capable of significantly preventing or delaying the oxidativedamage of substrates which are present at higher concentrations than the antioxidantsthemselves. Enzymatic antioxidants are perhaps the most well known and are largelyresponsible for maintaining a reducing intracellular environment in cells. These antioxidantsinclude superoxide dismutases (SODs), catalases and peroxidases.1.2.2 UR and transplantationI/R injury plays a significant role in endothelial dysfunction and the pathophysiologyof cAv.113- 118, 143 Then transplant organ is vulnerable to I/R injury induced by graft ischemiatime, quality of graft preservation during transport, hemodynamic status of the donor,catecholamines used for inotropic support, and reperfusion itself. 116 Three sequential phasesof graft ischemic time contribute to graft injury during transplantation: (1) the episode of22Chapter 1warm ischemia upon removal of the heart from the donor, (2) the cold ischemic intervalassociated with storage and preservation of the heart, and (3) the period of warm ischemiaduring engraftment. 114 Paradoxically, although reperfusion is required to restore tissueoxygenation, much of the damage that ensues during transplantation is associated with theoxidative burst that occurs during reperfusion. 114Compelling evidence supports a molecular and cellular basis for a causal relationshipbetween UR injury during transplantation and the onset and progression of CAV. 113' 143 URinjury to endothelial cells may provide the initial trigger for atherogenesis by stimulatingplatelet adhesion, release of growth factors, upregulation of MHC Class I and II expression,release of donor antigens, expression of adhesion molecules, and proliferation of vascularsmooth muscle cells. (Reviewed in 113-118, 143)Several experimental models using superoxide dismutase and antioxidants havedemonstrated the importance of ROS in the pathophysiology of UR injury. However, thedevelopment of effective treatments to alleviate reperfusion injury remains elusive.Furthermore, several candidate pathways have been proposed to produce ROS during URincluding mitochondria, NADPH oxidases, xanthine oxidase and eNOS. However, the datasupporting a role for these systems in UR injury remain inconclusive. For example, targeteddeletion of P47Ph07, an essential component of NADPH oxidase, abrogates NADPH-dependent superoxide generation in endothelial cells. However, UR studies in p47-null micereveal no significant difference in infarct size. 144 Similarly, xanthine oxidase inhibitors havefailed to protect against I/R 144 while eNOS may play a protective role.14523Chapter 1Recently, apoptosis repressor with caspase recruitment domain (CARD), describedin section 1.3, and cytochrome p450 2C enzymes, described in section 1.4, have been foundto play roles in myocardial I/R injury. 146' 147 The role of these proteins in vascular UR injuryand CAV are unknown.1.3 Apoptosis Repressor with Caspase Recruitment Domain (ARC)ARC, apoptosis repressor with caspase recruitment domain, was first identified byGabriel Nunez's group in 1998. 148 It is a 23 kDa protein with an N-terminal CARD domainand a C-terminal proline/glutamic acid rich domain. Its expression was originally thought tobe confined to terminally differentiated skeletal and cardiac muscle. 148 More recently, ARChas been shown to be expressed in cancer cells. 149150 Caspases and other CARD containingproteins are known to bind to one another through this domain. ARC was found to bind andinhibit caspase-2 and 8 indicating important implications in apoptosis. 148 ARC is also able toinhibit potassium efflux associated with apoptosis induction and cell shrinkage. 151Apoptosis is a tightly controlled form of cell death that is characterized by cellshrinkage, DNA fragmentation and membrane blebbing resulting in the packaging of the cellinto membrane-enclosed vesicles. These vesicles are then engulfed by surrounding`professional' (macrophages) or 'non-professional' phagocytes. Endothelial cell (EC) andSMC apoptosis and necrosis have been identified as important factors in the progression ofCAV. As described above, numerous factors such as oxidative stress, immune cells andcytokines participate in the induction of cell death in CAV. Therefore multiple death-inducing pathways must be inhibited in order to attenuate vascular damage. ARC is one of24Chapter 1the first known multifactorial apoptosis inhibitors and was shown to inhibit both apoptosisand necrosis in cardiac myoblasts. 152 Therefore, ARC may provide a unique method ofinhibiting the multiple apoptotic and necrotic pathways that are triggered in this disease.1.3.1 ARC in UR injuryARC is protective against ischemic injury and oxidative stress in cardiomyocyte andneuronal cells. Ekhterae et al:52 were the first to link ARC with protection against oxidativeinjury. They demonstrated that ARC overexpression was able to protect against hypoxia andre-oxygenation (H/R) induced caspase-3 activation, Poly (ADP-ribose) polymerase (PARP)cleavage and cytochrome c release. 152 Gustafsson et a1. 147 found that humanimmunodeficiency virus (HIV) transactivator of transcription (TAT)-fusion proteintransduction (described in section 2.1) of ARC was protective against oxidative injuryinduced by H202 in cultured embryonic myocytes and was protective against I/R injury inLangendorff perfused rat hearts. In the latter set of experiments, TAT-ARC transductionreduced both infarct size and creatine kinase (CK) release following I/R. 147 Chatterjee eta/. 153 found similar results using adenoviral transfer of ARC in a rabbit model of regionalcardiac ischemia. Treated animals maintained left ventricular geometry, had higher ejectionfractions and less border zone fractional shortening that control groups. 153 ARC-deficientmice demonstrate reduced contractile function, cardiac enlargement, and myocardial fibrosisfollowing aortic banding. 154 These mice also show increased infarct areas following I/R. 154Studies in hippocampal neurons showed that hypoxia downregulates ARC expression in thehippocampus and that overexpression of ARC protects against hypoxia-induced death in25Chapter 1these cells. 155 There are currently no data indicating whether or not ARC is protectiveagainst ischemic injury in the vasculature or whether that protection could reduce CAVdevelopment.1.4 Cytochrome p450 Enzymes (CYPsCYPs are membrane-bound, heme-containing terminal oxidases that are found inorganisms ranging from archaebacteria to humans. These enzymes are responsible for themetabolic 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 otherforeign substances in an oxygen and NADPH-dependent manner. The majority of CYPsisoforms are mono-oxygenases that catalyze the incorporation of a single atom of oxygeninto a substrate. CYPs are critical mediators of drug metabolism. Thus, considerable attentionhas been given to these enzymes by the pharmaceutical industry with respect to their role indrug-drug interactions, drug bioavailability and toxicity. 156There is substantial inter-individual variation in the activities of various CYPisoforms in humans resulting in differential metabolism, detoxification and/or clearance ofxenobiotics. Much of this inter-individual variability can be attributed to polymorphisms inCYP genes resulting in altered activity or expression of the encoded enzyme. However, CYPactivity is also heavily influenced by other factors such as drugs, hormones, development,•diet and cytolunes. 157 ' 158 Thus, both genetic and epigenetic components determine the abilityof an individual to metabolize a particular drug or toxic substance. To add furthercomplexity, the sequencing of the mouse, rat and human genomes has revealed substantial26Chapter 1differences in the CYP makeup of these animals. 159-16I For instance, the 2J subfamily, whichhas been shown to be abundantly expressed in the heart, has one member in humans, 4members in rats and 8 members in mice. 159 Furthermore, mice contain 84 different CYPisoforms versus only 63 isoforms in humans. 161 Thus, caution must be used when assessingdrug metabolism or the activation/deactivation of other toxins in rodents with respect totranslating this research to humans as rodents possess CYP isoforms that are not present inhumans and vice-versa. This may be one explanation as to why many therapeutics areeffective in mice, but fail in humans.The liver expresses the highest levels of CYP and plays a dominant role in the first-pass clearance of ingested xenobiotics and in the regulation of systemic levels of drugs andother chemicals. However, extra-hepatic tissues also possess CYP and contribute not only tofirst-pass clearance, but may also influence tissue burden of foreign compounds orbioavailability of therapeutics. 162 Many substances require CYP-mediated metabolicactivation to form toxicants or carcinogens. The reactive intermediates that are produced arefor the most part unstable and unlikely to be transported from the liver to other tissues toexert toxicity. Thus, chemical toxicity in extra-hepatic tissues may be regulated by CYP-mediated in situ metabolic activation in the target organ itself. 1621.4.1 CYP 2C enzymesCYP 2C enzymes are mono-oxygenases that catalyze the transfer of a single oxygenmolecule to their substrates. This process requires electron transfer from NADPH to27Chapter 102 + 2H+ 3 H20202- + NO  3 ON00-Figure 1.4 The CYP mono-oxygenase reaction cycleO2 is generated during the CYP reaction cycle when the electrons for the reduction of thecentral heme iron are transferred on the activated bound oxygen molecule. 0 2' is then readilyconverted to other ROS and RNS through reactions such as those shown for H202 andONOO-.28Chapter 1cytochrome 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 andrelease of ROS. 163 CYP mono-oxygenases produce superoxide during three stages of theirreaction cycle and can produce superoxide by NADPH consumption even in the absence ofsubstrate. 163 Substrate availability further increases the catalytic activity of CYPs and resultsin an increase in superoxide production.CYP 2C9 was mapped to chromosome 10 in humans 164, to chromosome 7 in miceand chromosome 1 in rats. 165 In the heart CYP 2C9 was found to be predominantly expressedon the right side, more specifically in the right ventricle and also in the vasculature. 166 Muchof what we know about CYP 2C9 comes from studies related to the metabolism of the manydrugs that it metabolizes. Tolbutamide, used in the treatment of type 2 diabetes, ismetabolized by CYP 2C9. An uncommon variant of the CYP 2C9 gene seems to beassociated with a reduced ability to metabolize tolbutamide. This same variant, CYP 2C9*3in which isoleucine at position 359 is mutated to leucine, is also associated with both reducedclearance 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 thestructure 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 populationcompared to the black population. 169 Two CYP 2C9 variants have been identified as poormetabolizers of warfarin, CYP 2C9*2 (arg144—>cys) and CYP 2C9*3 described above. In aretrospective cohort study of patients being treated with warfarin, individuals with poormetabolizing CYP 2C9 variants were associated with an increased risk of bleeding events. 17°29Chapter 1Although 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 havedetected an isoform corresponding to a similar sized protein in rat heart protein extracts usingan antibody for human CYP 2C9 and have demonstrated that CYP 2C9 inhibitors reducepost-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 liverpreparation5. 1731.4.1.1 CYP 2C in vascular homeostasisAlthough the majority of CYP are most abundantly expressed in the liver, CYP arealso expressed in extra-hepatic tissues including the heart and the vasculature. 174 The humanAA-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 Theseepoxygenases generate epoxyeicosatrienoic acids (EETs), ROS, and other products.Vascular tone and homeostasis is modulated by numerous vasoactive signals andcompounds produced by the autonomic nerves, the tissue, and the endothelium. 174Vasodilators include vascular flow, the well-known autacoids, NO  and prostacyclin (PGI2),and several less well characterized receptor-mediated agonists. 174 NO/PGI2-independentpathways make a significant contribution to vasodilation, particularly in the renal, mesenteric,and coronary arteries. 174 Endothelium-derived hyperpolarizing factor (EDHF) is an agonistthat causes the hyperpolarization of endothelial and smooth muscle cells though both Na-K-ATPase and calcium-dependent K ± channels.' 75' 176 CYP have been linked to EDHF activity30Chapter 1because CYP specific inhibitors, such as 6 (2-proparglyoxyphenyl) hexanamide, can preventNO/PGI2-independent vasodilation. 177 Furthermore, an antisense approach against the CYP2C family was able to demonstrate an attenuation of bradykinin-induced EDHF-mediatedvascular responses without affecting NO-mediated vascular responses. 178 Moreover,sulfaphenazole, a selective inhibitor of CYP 2C9, was able to inhibit EDHF-mediatedvasodilation in porcine coronary arteries. 179 This research implicates CYP 2C9 as a putativeEDHF synthase and 1 1,1 2-EET as the putative EDHF.CYP products such as EETs and hydroecosotraenoic acids (HETEs) as well as theirdegradation products have been associated with both the induction and inhibition ofvasodilation. For example, CYP 2J2 is localized to the endothelium of large and smallcoronary arteries and is able to generate not only EET from AA, but alsoepoxyeicosaquatraenoic acids from eicosapentaenoic acid. 18° Both EET andepoxyeicosaquatraenoic acid are known dilators of the microvasculature. 18° The diol productsof EETs, dihydroxyeicosatrienoic acids, can be taken up by ECs and cardiac myocytes andincorporated into phosphatidylcholine, phosphatidylinositol, and to a lesser extent otherphospholipids. 181 Even when EETs are released into the extracellular environment they arebelieved to incorporate into circulating lipoproteins through esterification. 182 It ishypothesized that the incorporation of EETs into phospholipids serves as a means of storingthese molecules, but it is not known if EETs are also active in this form. 174 Unfortunately, theeffects of EETs on vasodilation have typically been measured in the presence of inhibitors ofNO-dependent vasodilation. This is a concern because NO  is an inhibitor of CYP. For thatreason, it is unknown how much of an effect EETs have on vasodilation in the presence ofNO . This is further complicated because CYP also generate ROS during their reaction cycle3 1Chapter 1as electrons are transferred from the central heme iron to the activated bound oxygenmolecule. 179 In fact, CYP make a significant contribution to the cellular production of ROSsuch as^H202 and hydroxyl radicals. 179 Through the production of free radicals, CYPmay also contribute to vascular homeostasis because ROS are known participants in themaintenance of vascular tone and homeostasis. 183 Unlike the EET products of CYP, ROS areimplicated in the inhibition of NO-mediated relaxation. 027' reacts with NO  to form ON00-thus reducing the bioavailability and vasoactivity of NO . 1791.4.1.2 CYP 2C in YR injuryYasar et al. (2003) 184 examined correlations between genetic variants of CYP 2C8and 2C9 and risk of acute MI. An increased risk of acute MI has been associated with thegenetic CYP variants CYP 2C9*2 and *3 in female patients, and CYP 2C8*3 in both malesand females. 184 These variants have reduced activity compared to their wild-typecounterparts. 185, 186 Recent studies from our group suggest that the rat CYP 2C9-equivalent isan important mediator of I/R injury. 146 In the latter study, several CYP inhibitors were testedfor their ability to protect against cardiac YR injury. Three structurally-unrelated CYP mono-oxygenase 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 orhuman CYP 2C9. Thus, it became apparent that CYP 2C6/9 may be a key player in cardiacI/R injury. In rat hearts perfused in Langendorff mode, the CYP inhibitors reduced infarct32Chapter 1size, ROS production and CK release compared to that of controls. 146 Similar results werefound in a rabbit model of left anterior descending coronary artery constriction. CYP 2C9inhibitors also increased post-ischemic coronary flow suggesting that increased vasodilationand/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 riskfactors for heart attacks, such as tobacco smoke and cocaine, are potent inducers of CYPs inthe 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-inducedcardiovascular disease than cancer, the mechanism by which smoking contributes tocardiovascular disease is poorly understood. However, there is evidence to suggest CYPmight be responsible. The role of CYP in smoking-related cancer is well-established andrecent findings indicate that certain CYP isoforms are involved in atherogenesis.Polymorphisms in CYP 1A1, one of the key detoxifying enzymes catabolizing cigarettesmoke-derived toxins, are associated with smoking-induced atherogenesis. CYP 1A1polymorphisms have been associated with susceptibility to severe coronary artery diseaseand type 2 diabetes in smokers. 189 Further evidence for a role of CYP in MI stems fromstudies of cocaine-induced heart attacks. Cocaine can induce acute MI in young adults 190 andhas been reported to be a potent inducer of CYP in cardiac tissues. 193 Conversely, althoughseveral mechanisms have been forwarded to explain the cardioprotective effects ofpolyphenolic compounds found in red wine and other foods 191 ' 194-197 , it is of interest to notethat these substances are also known CYP inhibitors. 191' 194-197 In summary, there is powerfuland accumulating indirect evidence supporting a role for CYP in tissue-specific cytotoxicityand cardiovascular disease.33Chapter 11.4.1.3 CYP 2C in atheromatous diseaseThum and Borlak 198 have implicated oxidized low density lipoprotein (oxLDL) in thedownregulation of CYP mono-oxygenases in coronary arterial endothelial cells. This studylinked 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 Asignificant decrease in the expression of CYP 1A1, 2A6/7, 2B6/7, 2C8, 2C9, 2E1, and 2J2was detected in coronary arterial endothelial cells treated with oxLDL, but not in cells treatedwith normal LDL. 198 Fichtlscherer et al." showed that CYP 2C9 inhibition viasulfaphenazole is associated with increased endothelium-dependent vasodilation in humanpatients with coronary artery disease. 20° This effect was attributed to a decrease in ROSproduction by CYPs, as well as a consequent increase in NO  bioavailability and NO-mediated vasodilation. This work suggests inhibition of CYP 2C9 as a possible therapeuticintervention to maintain blood flow and protect against ischemic damage in patients withestablished coronary artery disease."1.4.1.4 CYP 2C in other cardiovascular diseasesCYPs have been speculated to play a significant role, both in the onset of and theprotection against a broad spectrum of cardiovascular diseases. While CYPs have beenstudied extensively in drug metabolism in the liver, studies into their roles in xenobioticmetabolism and the production of biologically active metabolites and toxins in the heartrequires further elucidation. CYPs have recently been implicated in the induction of34Chapter 1angiogenesis. Furthermore, endothelial cell proliferation, associated with angiogenesis, islinked with CYP 2C9 expression. Human umbilical vein endothelial cells infected withadenovirus to overexpress CYP 2C9 demonstrated a 50% increase in proliferation overantisense infected cells as well as a 3-fold increase in cyclin D1 expression. 201 This increasedendothelial proliferation was prevented with the addition of a CYP 2C9 specific inhibitorsulfaphenazole. 201 Administration of 11,12-EET to chick choriollantoic membranes was ableto induce angiogenesis to a similar degree as known pro-angiogenic factors such asendothelial growth factor (EGF) and vascular endothelial growth factor. Again, the inductionof angiogenesis by 11,12-EET was inhibited using AG1478 as well as an EGF neutralizingantibody. Similar experiments in a human lung microvascular cell lines showed a more than25% 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 infusedsubcutaneously on the dorsal midline of a rat. Angiogenesis was subsequently measured asindicated by haemoglobin content and by immunostaining of platelet endothelial celladhesion molecule-1 (PECAM). After one week 14,15-EET treated Matrigel showed a 1.6-fold increase in haemoglobin over control as well as positive PECAM staining. 202 Therelative contributions of 11,12- and 14,15-EET and the mechanism of EGF receptorinvolvement in CYP 2C9 induced angiogenesis are currently unknown and require furtherexperimentation to be fully elucidated. CYP 4A1 was also shown to induce angiogenesis inrenal interlobar arteries in a smooth-muscle cell dependent manner. 203Hypertension, or high blood pressure, is a leading cause of death, MI, stroke, andother illness in North America. It is typically asymptomatic and the great majority ofpatients have essential hypertension, in which the cause of blood pressure elevation is35Chapter 1unknown. 2°4 Hypertension is largely regulated by the cardiac output of the heart, the systemicresistance controlled by blood vessel tone, and the intravascular tone regulated by thekidneys. CYPs have often been considered when treating hypertensive patients due to theirinteractions with anti-hypertensive drugs such as candesartan 205, warfarin206, phenytoin, andtolbutamide207. 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 beenlinked to the development of hypertension. There have been several reports, however,describing conflicting roles for CYP in hypertension. Fenofibrate, a drug known to induceexpression of CYP 4A and elevate production of 20-HE1E, has been shown to reduce bloodpressure in stroke prone spontaneously hypertensive rats. 208 On the other hand, the use of17-octadecynoic acid, an inhibitor of EET and 20-HE'1E production, was also able to reduceblood pressure in Lyon hypertensive rats. 209Single nucleotide polymorphisms (SNP) of CYP 2C9 were studied to determine if itwas possible to predict the efficacy of treatment with irbesartan, a drug used to treathypertension, with SNP information. 210 The results indicated that the rate of irbesartanmetabolism is indicative of the CYP 2C9 genotype expressed, providing a valuable use forgenotyping before treatment of hypertension.1.5 Arachidonic Acid (AA) MetabolismAA is metabolized by three major pathways; the cyclooxygenase (COX) pathway, thelipoxygenase (LOX) pathway, and the CYP epoxygenase pathway. These pathways areshown in Figure 1.5.36Cytochrome p450EpoxygenasesChapter 1Arachidonic AcidCyclooxygenases LipoxygenasesDHETEs, HETEs, EETs, ROSLeukotriene,HPETE, DEAProstaglandins, Prostacyclin,Thromboxane A2Figure 1.5: Overview of the three pathways of arachidonic acid metabolism.AA is metabolized by three main pathways; the cyclooxygenase pathway, the lipoxygenasepathway and the cytochrome p450 epoxygenase pathway. The main products of thesepathways are shown as described in 211 ' 212.37Chapter 11.5.1 AA metabolism by cyclooxygenaseThe 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 theylead to formation of PGH. There are three types of COX. COX-1 and COX-3 areconstitutively expressed and are present in the stomach, kidney and thrombocytes and thebrain, respectively. COX-2 is the inducible form of the enzyme and is present in multipletissues including the heart and vasculature. However, COX-2 is thought to be constitutivelyexpressed in some tissues including gastric tissues and endothelial cells. 213 Initially COXoxidizes AA into the endoperoxides PGH2 and PGB2 which are precursors of theprostaglandins PGE2, PGF2, PGI2 and of thromboxanes (TX).213 The production of thesemetabolites differs in different tissues with TX formation dominating in blood platelets 214and prostaglandin and prostacyclin formation dominating in vascular cells.1.5.2 AA metabolism by lipoxygenaseThe LOX are dioxygenases that metabolize AA into HPETEs(hydroperoxyeicosatraenoic acids) and DEA (dihydroxyeicosatraenoic acid). These productsare then converted to HETEs, leukotrienes, and lipoxins by peroxidases, hydrase andglutathione S-transferase, and lipoxygenases, respectively. There are two main LOXenzymes 5-LOX and 12-LOX, defined by the carbon atom on which the oxygen is fixed. 5-LOX is present in many cell types and leads to the formation of leukotrienes (LT) A4, B4and subsequently through modifications LTC4, LTD4 and LTE4. 12-LOX is more restrictedin its expression being present in skin, thrombocytes and some tumours.38Chapter 11.5.3 AA metabolism by CYPsThe epoxygenase pathway employs CYP epoxygenases in the formation of EETs andHETEs.211 CYP 2C are the primary epoxygenases involved in AA metabolism by the thirdpathway.215 Unfortunately, in addition to the production of EETs and HETEs, CYP 2C alsomake a significant contribution to the cellular production of ROS such as 02', H202 andhydroxyl radicals. 179' 216AA metabolism is increased during myocardial ischemia and to an even greaterdegree during reperfusion. 212 The COX and CYP pathways are largely responsible for theincrease in AA metabolism during I/R. This increase is the result of an increase inintracellular calcium levels during FR which activates phospholipase A2 (PLA2). PLA2catalyses the hydrolysis of AA from membrane phospholipids thus increasing theconcentration of free AA in the cytosol.217-220 AA metabolism, on the whole, is detrimentalduring UR. Several studies, in multiple cell types, have demonstrated that inhibition of AAmetabolism 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 andAA causes CYP-mediated superoxide production in isolated renal microsomes. 216 We havepreviously demonstrated that CYP 2C contributes to vascular and cardiac post-ischemic 02 -*production, 146, 227 likely as a result of increased AA metabolism.39Chapter 11.5.4 AA metabolites and cardiovascular diseaseCOX-2 is constitutively expressed endothelial cells, however, COX-2 levels are alsoknown to be induced by cytokines, growth factors, lipopolysaccharides, prostanoids andsubstrates.213 COX-2 is also known to be bound to PGI2 synthase in endothelial cells resultingin PGI2 being the predominant AA metabolite in these cells. PGI2 is anti-thrombotic andvasodilatory and plays a central role in vascular homeostasis. 228Coxibs, selective COX-2 inhibitors, have been associated with increasedcardiovascular events. The first studies were related to rofecoxib, Vioxx, and eventually ledto its withdrawal from the market. The VIGOR study, of rofecoxib, showed a nearly 5-foldincreased risk of myocardial infarction in those patients that received rofecoxib 229 and acorrelation between myocardial infarction and rofecoxib was also found by the APPROVe(Adenomatous Polyp PRevention On Vioxx) study of rofecoxib. 38 Studies related tocelecoxib and paracoxib/valdecoxib have also shown an association with increasedcardiovascular risks.23°-232 These later studies resulted in warnings related to increasedcardiovascular risks for patients taking celecoxib and the voluntary withdrawal orparacoxib/valdecoxib from the market. Although coxibs have been associated with increasedrisk of cardiovascular events, and it is likely that decreased PGI2 synthesis contributes tothese events, the underlying mechanisms and other contributing factors have not been fullyexamined.During ischemia increased intracellular calcium levels induces the activation of PLA2and the subsequent hydrolysis of AA from membrane phospholipids. 217-220 Several AAmetabolites, including COX-2 derived PGI2, are known to be cardioprotective. The negative40Chapter 1cardiovascular effects of COX-2 inhibitors have been largely attributed to decreasedprostacyclin production. However, the overall metabolism of AA during I/R has beenimplicated 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 prostacyclinproduction upstream of COX-2 at the point of AA hydrolysis does not have the samenegative cardiovascular effects. This implies that the effects of COX-2 on the cardiovascularsystem are more complex than inhibition of PGI2 production alone.CYPs, primarily the CYP 2C epoxygenases, are often referred to as the third pathwayof AA metabolism (LOX and COX being the other two). We have previously demonstratedthat CYP 2C contributes to vascular dysfunction and myocardial injury following I/R. In thelatter study, inhibition of CYP 2C by sulfaphenazole reduced myocardial infarction by nearly60%. CYP 2C undergoes substrate-induced activation. As AA can be metabolized by one of3 possible mechanisms, it is logical that if one of these pathways were blocked, that this mayresult 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 epoxygenaseshas not been examined.1.6 Thesis objectives and hypothesesThe overall objective of this thesis is to examine potential mechanisms to reduce peri-transplant ischemic injury in the vasculature and to assess the relationship between this formof injury and the development of CAV.41Chapter 1To this end we examined the potential of the anti-apoptotic protein ARC to preventoxidant cell death in the vasculature. We hypothesized that inhibition of apoptotic andnecrotic cell death in the donor heart through increased ARC protein levels would attenuateI/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-transplantischemic injury and CAV development. We hypothesized that CYP 2C enzymes play a keyrole in the pathogenesis of CAV through the production of reactive oxygen species thatcontribute to inflammation, endothelial damage and dysfunction. Our initial examinations,described in Chapter 3, determined the influence of the rodent CYP 2C9-equivalent on I/R-mediated vascular dysfunction in coronary arteries isolated from Langendorff-perfused hearts.We then assessed the contribution of rodent CYP 2C on peri-transplant ischemic injury andCAV using a rat heterotopic heart transplant model of chronic rejection. Results from thesestudies are described in Chapter 4. Finally, we assessed the effects of CYP 2C9 onhypoxia/re-oxygenation (H/R) induced cell death in EC and SMC and examined questionsrelated to altered oxidative stress and AA metabolism following CYP 2C9 inhibition.Results described in this thesis demonstrate that ARC does not have similar protectiveeffects against oxidant induced injury in vascular cells as it does in myocytes. Further weserendipitously discovered a novel role for ARC in myogenic differentiation. We havedemonstrated that CYP 2C contributes to endothelium-dependent vascular dysfunction andvascular ROS generation following I/R. Inhibition of CYP 2C during cardiac transplantationwas found to be protective against CAV development and that expression of CYP 2C9increases cell death and may alter AA metabolism in cultured human ECs.42Chapter 11.7 Bibliography1. Squifflet JP. From leg transplantation by St Cosmas and St Damian to the modern era.Acta Chir Belg. 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Effects of resveratrol, a flavinoid found in red wine, on infarctsize in an experimental model of ischemia/reperfusion. J Stud Alcohol. Nov2001;62(6):730-735.196. Sato M, Maulik N, Das DK. Cardioprotection with alcohol: role of both alcohol andpolyphenolic antioxidants. Ann N Y Acacl Sci. May 2002;957:122-135.197. Das DK, Sato M, Ray PS, et al. Cardioprotection of red wine: role of polyphenolicantioxidants. Drugs Exp Clin Res. 1999 ; 25 (2-3): 115-120.198. Thum T, Borlak J. Mechanistic role of cytochrome P450 monooxygenases inoxidized low-density lipoprotein-induced vascular injury: therapy through LOX-1receptor antagonism? Circ Res. Jan 9 2004;94(1):e1-13.199. Morel Y, Barouki R. Down-regulation of cytochrome P450 1A1 gene promoter byoxidative stress. Critical contribution of nuclear factor 1. J Biol Chem. Oct 91998;273(41):26969-26976.54Chapter 1200. Fichtlscherer S, Dimmeler S, Breuer S, et al. Inhibition of cytochrome P450 2C9improves endothelium-dependent, nitric oxide-mediated vasodilatation in patientswith coronary artery disease. Circulation. Jan 20 2004;109(2):178-183.201. Michaelis UR, Fisslthaler B, Medhora M, et al. Cytochrome P450 2C9-derivedepoxyeicosatrienoic acids induce angiogenesis via cross-talk with the epidermalgrowth factor receptor (EGFR). Faseb J. Apr 2003;17(6):770-772.202. Medhora M, Daniels J, Mundey K, et al. Epoxygenase-driven angiogenesis in humanlung microvascular endothelial cells. Am J Physiol Heart Circ Physiol. Jan2003;284(1):H215-224.203. Jiang M, Mezentsev A, Kemp R, et al. Smooth muscle--specific expression ofCYP4A1 induces endothelial sprouting in renal arterial microvessels. Circ Res. Feb 62004;94(2):167-174.204. Malhotra RWG, Lilly L.S. Hypertension In Pathophysiology of Heart Disease: Acollaborative Project of Medical Students and Faculty. Baltimore, Maryland.:Lippincott Williams & Wilkins; 2003.205. Uchida S, Watanabe H, Nishio S, et al. Altered pharmacokinetics and excessivehypotensive effect of candesartan in a patient with the CYP2C91/3 genotype. ClinPharmacol Ther. Nov 2003;74(5):505-508.206. Zhu GJ, Yu YN, Li X, et al. Cloning of cytochrome P-450 2C9 cDNA from humanliver and its expression in CHL cells. World J Gastroenterol. Apr 2002;8(2):318-322.207. Shi MM. Enabling large-scale pharmacogenetic studies by high-throughput mutationdetection and genotyping technologies. Clin Chem. Feb 2001;47(2):164-172.208. Shatara RK, Quest DW, Wilson TW. Fenofibrate lowers blood pressure in twogenetic models of hypertension. Can J Physiol Pharmacol. May 2000;78(5):367-371.209. Messer-Letienne I, Bernard N, Roman 12.1, et al. 20-Hydroxyeicosatetraenoic acid andrenal function in Lyon hypertensive rats. Eur J Pharmacol. Aug 13 1999;378(3):291-297.210. Hallberg P, Karlsson J, Kurland L, et al. The CYP2C9 genotype predicts the bloodpressure response to irbesartan: results from the Swedish Irbesartan Left VentricularHypertrophy Investigation vs Atenolol (SILVHIA) trial. J Hypertens. Oct2002;20(10): 2089-2093.211. Bylund J, Ericsson J, Oliw EH. Analysis of cytochrome P450 metabolites ofarachidonic and linoleic acids by liquid chromatography-mass spectrometry with iontrap MS. Anal Biochem. Dec 1 1998;265(1):55-68.212. Hendrickson SC, St Louis JD, Lowe JE, et al. Free fatty acid metabolism duringmyocardial ischemia and reperfusion. Mol Cell Biochem. Jan 1997;166(1-2):85-94.213. Krotz F, Schiele TM, Klauss V, et al. Selective COX-2 inhibitors and risk ofmyocardial infarction. J Vasc Res. Jul-Aug 2005;42(4):312-324.214. Helliwell RJ, Adams LF, Mitchell MD. Prostaglandin synthases: recent developmentsand a novel hypothesis. Prostaglandins Leukot Essent Fatty Acids. Feb2004;70(2):101-113.215. Luo G, Zeldin DC, Blaisdell JA, et al. Cloning and expression of murine CYP2Cs andtheir ability to metabolize arachidonic acid. Arch Biochem Biophys. Sep 11998;357(1):45-57.216. Fulton D, McGiff JC, Wolin MS, et al. Evidence against a cytochrome P450-derivedreactive oxygen species as the mediator of the nitric oxide-independent vasodilator55Chapter 1effect of bradykinin in the perfused heart of the rat. J Pharmacol Exp Ther. Feb1997;280(2):702-709.217. Freyss-Beguin M, Millanvoye-van Brussel E, Duval D. Effect of oxygen deprivationon metabolism of arachidonic acid by cultures of rat heart cells. Am J Physiol. Aug1989;257(2 Pt 2):H444-451.218. Leong LL, Sturm MJ, Ismail Y, et al. Plasma phospholipase A2 activity in clinicalacute myocardial infarction. Clin Exp Pharmacol Physiol. Feb 1992 ;19(2): 113-118.219. Van der Vusse GJ, Reneman RS, van Bilsen M. Accumulation of arachidonic acid inischemic/reperfused cardiac tissue: possible causes and consequences. ProstaglandinsLeukot Essent Fatty Acids. Jul 1997;57(1):85-93.220. Williams SD, Gottlieb RA. Inhibition of mitochondrial calcium-independentphospholipase A2 (iPLA2) attenuates mitochondrial phospholipid loss and iscardioprotective. Biochem J. Feb 15 2002;362(Pt 1):23-32.221. Ogata K, Jin MB, Taniguchi M, et al. Attenuation of ischemia and reperfusion injuryof canine livers by inhibition of type II phospholipase A2 with LY329722.Transplantation. Apr 27 2001;71(8):1040-1046.222. Sargent CA, Vesterqvist 0, McCullough JR, et al. Effect of the phospholipase A2inhibitors quinacrine and 7,7-dimethyleicosadienoic acid in isolated globally ischemicrat hearts. J Pharmacol Exp Ther. Sep 1992;262(3):1161-1167.223. Czerniecki BJ, Witz G. Arachidonic acid potentiates superoxide anion radicalproduction by murine peritoneal macrophages stimulated with tumor promoters.Carcinogenesis. Oct 1989 ; 10(10): 1769-1775.224. Mayer AM, Brenic S, Stocker R, et al. Modulation of superoxide generation in invivo lipopolysaccharide-primed rat alveolar macrophages by arachidonic acid andinhibitors of protein kinase C, phospholipase A2, protein serine-threoninephosphatase(s), protein tyrosine kinase(s) and phosphatase(s). J Pharmacol Exp Ther.Jul 1995;274(1):427-436.225. Mayer AM, Spitzer JA. Modulation of superoxide generation in in vivolipopolysaccharide-primed Kupffer cells by staurosporine, okadaic acid, manoalide,arachidonic acid, genistein and sodium orthovanadate. J Pharmacol Exp Ther. Jan1994;268(1):238-247.226. Toborek M, Malecki A, Garrido R, et al. Arachidonic acid-induced oxidative injury tocultured spinal cord neurons. J Neurochem. Aug 1999;73(2):684-692.227. Hunter AL, Bai N, Laher I, et al. Cytochrome p450 2C inhibition reduces post-ischemic vascular dysfunction. Vascul Pharmacol. Oct 2005;43(4):213-219.228. Liou JY, Shyue SK, Tsai MJ, et al. Colocalization of prostacyclin synthase withprostaglandin H synthase-1 (PGHS-1) but not phorbol ester-induced PGHS-2 incultured endothelial cells. J Biol Chem. May 19 2000;275(20):15314-15320.229. Bombardier C, Laine L, Reicin A, et al. Comparison of upper gastrointestinal toxicityof 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 1528.230. Nussmeier NA, Whelton AA, Brown MT, et al. Complications of the COX-2inhibitors parecoxib and valdecoxib after cardiac surgery. N Engl J Med. Mar 172005;352(11):1081-1091.56Chapter 1231. Ott E, Nussmeier NA, Duke PC, et al. Efficacy and safety of the cyclooxygenase 2inhibitors parecoxib and valdecoxib in patients undergoing coronary artery bypasssurgery. J Thorac Cardiovasc Surg. Jun 2003;125(6):1481-1492.232. Solomon SD, McMurray JJ, Pfeffer MA, et al. Cardiovascular risk associated withcelecoxib in a clinical trial for colorectal adenoma prevention. N Engl J Med. Mar 172005;352(11):1071-1080.57Chapter 2: Apoptosis Repressor with CaspaseRecruitment Domain in Vascular Cell Death andMyogenic Differentiation )2.1 IntroductionApoptosis is a tightly controlled form of cell death that is characterized by cellshrinkage, DNA fragmentation and membrane blebbing resulting in the packaging of the cellinto membrane-enclosed vesicles. These vesicles are then engulfed by surrounding`professional' (macrophages) or 'non-professionar phagocytes. Endothelial cell and smoothmuscle cell apoptosis and necrosis have been identified as important factors in theprogression 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 insection 1.1.3.2. Therefore, multiple death inducing pathways must be inhibited in order toattenuate vascular damage.ARC was originally discovered as a caspase-2 and -8-interacting, anti-apoptotic proteinthat is expressed primarily in the heart and skeletal muscle. 1 ARC is capable of preventingboth 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 ofintersecting with both the intrinsic and extrinsic apoptotic pathways. Overexpression of ARCinhibits ischemia-induced apoptosis in cardiomyoblast H9c2 cells by preventing58A 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 myocytedifferentiation by apoptosis repressor with caspase recruitment domain (ARC). FEBS Lett.581(5):879-84.Chapter 2mitochondrial cytochrome c release4 and, in Langendorff-perfused rat hearts, TAT-transduction of ARC was shown to significantly reduce infarct size following ischemia andreperfusion. 5ARCs ability to inhibit both apoptotic and necrotic forms of cell death whentransfected into cardiac myoblasts 4, may provide a unique method of inhibiting the multipleapoptotic and necrotic pathways involved in ischemic injury and CAV. However, little isknown about the expression or activity of ARC in EC or SMCs.HIV TAT-mediated protein transduction has been developed as a highly efficientmethod of transducing biologically active proteins into cells and tissues in vivo. Thetechnology requires the synthesis of a fusion protein, linking the arginine-rich, 11 amino acidTAT protein transduction domain, to the protein of interest using a bacterial expressionvector 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 previouslybeen shown to be detectable and functional in all tissues, including the heart. 6 Proteintransduction occurs with nearly equivalent concentrations in all cells in the transducedpopulation within 15 min, in a dose-dependent manner.7-92.2 AimAs ARC has previously been shown to protect against cardiac I/R injury5 and URinjury is associated with the development of cardiac allograft vasculopathy (discussed insection 1.2.2), we hypothesized that ARC may be protective against peri-transplant ischemic59Chapter 2injury and prevent the development of cardiac allograft vasculopathy. The aim of this chapteris to explore the potential for ARC to protect against oxidative damage in cardiovascular celltypes in culture as a marker of ischemic injury. In this study, we examined the nativeexpression of ARC in EC, SMC and cardiomyocytes. We examined the effects of alteredARC levels on protection against oxidative damage. In the course of these experiments wehave found compelling evidence that ARC inhibits myoblast differentiation. We examinedalterations in native ARC expression following the induction of differentiation as well as theeffect of ARC overexpression on muscle cell differentiation using H9c2 rat myoblasts as amodel. 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 regulatoryprotein ARC in myoblast differentiation2.3 Materials and Methods2.3.1 Cell culturePooled human umbilical venous endothelial cells (HUVECs) and human coronaryartery smooth muscle cells (HCASMCs) were obtained from Cambrex (Baltimore, MD).HUVECs were cultured in complete endothelial growth medium (EGM: endothelial basalmedium supplemented with 0.4% bovine brain extract, 0.1% human endothelial growthfactor (hEGF), 0.1% hydrocortisone and 0.1% gentomycin-amphotericin B (GA-1000);Cambrex) plus 5% foetal bovine serum (FBS, Invitrogen). HCASMCs were cultured incomplete smooth muscle growth medium (SmGM: smooth muscle basal medium60Chapter 2supplemented with 0.1% insulin, 0.5% human foetal growth factor B, 0.1% GA-1000 and0.1% hEGF; Cambrex) plus 5% FBS. The rat embryonic cardiac cell line H9c2 was obtainedfrom the American Type Culture Collection (Manassas, VA). They were grown andmaintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetalcalf serum (FCS) up to passage 23. All cells were cultured using sterile technique.2.3.2 RNA extraction and reverse transcriptase (RT)-PCRRNA was extracted from cultured HUVEC and HCASMCs to assess native ARCexpression. A minimum of 2.5 x 10 6 cells were trypsinized and collected and total RNA wasextracted 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 1volume of 70% ethanol was added. Samples were then centrifuged through the RNeasy minicolumn for 15 s at 10,000 rpm. The column was washed once with buffer RW1 and twicewith 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 RT-PCR. Purified RNA was subjected to DNase treatment to remove contaminating genomicDNA. Five micrograms of RNA was combined with 1X DNase I buffer, 5 mM MgC12, 1 mMdNTPs, 1X RNase inhibitor and DNase I. Samples were run at 37°C for 45 min, 99°C for 7min and then cooled to 4°C. Following DNase treatment, samples were treated with Qiagenpre-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: 1XPCR buffer, 1 mM MgC12, 2.5 U/100µ1 Taq, 200 dNTPs, 1.0 ILiM primers. Primer61Chapter 2sequences 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 gelpre-stained with ethidium bromide. Gels were imaged using the Strategene EagleEye IIultraviolet imager (Stratagene, La Jolla, CA).23.3 Cell lysis and Western blottingCells were washed two times with ice cold PBS and lysed in CellLytic M lysis buffercontaining protease inhibitor cocktail (Sigma-Aldrich, Oakville, ON). Protein concentrationswere 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 CoomassieBrilliant 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 gelelectrophoresis and then transferred to nitrocellulose membranes. After blocking with 5%skim milk, the membranes were incubated for 1 h with primary antibodies (1:1000 anti-myogenin antibody and 1:200 anti-skeleton muscle troponin T antibody (Santa CruzBiotechnology, 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 1:2000IRDye700Tm—conjugated secondary antibodies (Rockland Inc. Gilbertsville, PA). Proteinexpression was detected by using the Odyssey Infrared Imaging System from LI-CORBiosciences (Lincoln, NE).62Chapter 22.3.4 TAT protein expression, purification and Texas red stainingBL21(DE3)pLysS bacteria containing either the pTAT-HA-hARC plasmid or thepTAT-HA-P-gal plasmid were prepared as previously described5 and kindly provided by Dr.Roberta Gottlieb. The plasmids are modified from the pTAT-HA vector originallydeveloped 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 ampicillinresistance, a T7 promoter, 6x-Histadine (His) and hemagglutinin (HA) tags, and an N-terminal TAT peptide fusion cassette.Frozen glycerol stock cultures were transferred to Luria broth (LB) containingampicillin (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. Bacteriawere centrifuged at 5000 rpm for 15 min, washed once with PBS, and resuspended in BufferZ (8 M Urea, 100 mM NaCl, 20 mM HEPES pH 8.0) containing 20 mM imidazole. Bacterialsolutions were then sonicated on ice 3 times for 15 s pulses with 30 s on ice in betweenpulses and then centrifuged at 11700 rpm for 30 min. Supernatants were collected.Nickle-nitrilotriacetic acid (Ni-NTA) absorbent columns were used to purify the6xHis tagged proteins. Ni-NTA is a tetradentate chelating adsorbent which occupies four ofthe six ligand binding sites in the coordination sphere of the nickel ion, leaving two sites freeto interact with the 6xHis tag. The NTA is able to stably bind metal ions and retain themunder stringent wash conditions. The theoretical capability of this technique allowspurification 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) were63pRSETA,B.02.9 kbChapter 2 - -0^°^CS) _C^o —z 0. ct^co in ccYGRKKRRQRRRBS-ATG-His6—TAT— HA- MCS term/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 plasmidproduces a 6xHis, HA tagged TAT-fusion protein.NT7 ./64Chapter 2prepared by adding 10 ml of resuspended Ni-NTA resin and allowing excess fluid to runthrough by gravity. Columns were then pre-equilibrated with 50 ml of Buffer Z + 20 mMimidazole followed by the supernatants prepared above. Columns were then washed with 10bed volumes (2 x 25 ml) of Buffer Z + 10 mM imidazole. TAT-fusion proteins were theneluted by adding 10 ml of 250 mM imidazole in Buffer Z. Elution fractions were analyzedby 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), weredrained and equilibrated with 25 ml of sterile PBS. Elution fractions were then added andsamples 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 wasmeasured using the Bio-Rad modified Bradford assay described in section 2.2.3.To assess subcellular localization some TAT-fusion protein preparations were stainedwith Texas red succinimidyl ester (Molecular Probes, Eugene, OR). Texas red (12 mM inDMSO) was added at a molar ratio of dye to protein of 5:1 in 0.1 M bicarbonate buffer (pH8.3). 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 ofthe dye. Protein-dye mixtures were then desalted using PD-10 columns as described above.2.3.5 TAT-fusion protein transduction and detectionCells were grown to 70-90% confluency in complete media with 5% FBS. Media wasremoved and cells were washed with Dulbecco's PBS (DPBS). Media was then replaced with65Chapter 2serum free basal media. TAT-fusion proteins were added between 0-1 1,IM and wereincubated for 1 h at 37°C. Cells were then washed twice with DPBS and were then utilizedfor further experimentation. To assess levels of TAT-fusion protein transduction we utilizedproteins pre-stained with Texas Red. Following transduction media was replaced with phenolred free media and cells were imaged using the fluorescent microscope (595-605 nmexcitation, 615 nm emission).2.3.6 Cell viabilityHUVECs 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 asdescribed in section 2.2.5. Cells were then treated with 0.6 mM H202 for 4 h. Viability wasassessed using the CellTiter96TM AQueous Assay (MTS) (Promega, Madison, WI). MTS is acolorimetric assay involving a cell permeable novel tetrazolium compound [344,5-dimethylthiazol-2-y1)-5-(3-carboxymethoxypheny1)-2-(4-sulfopheny1)-2H-tetrazolium] andan electron coupling reagent (phenazine methosulfate) PMS. Upon entry in to viable cells,MTS is bioreduced to an aqueous soluble formazan product by dehydrogenase enzymes. Thesoluble formazan product is proportional to the number of viable cells and can be measuredspectrophoretically due to its absorbance at 490 nm. MTS was protected from light and wasadded 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 GENiosRainbow 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 measuredin triplicate.66Chapter 2H9c2-Neo or ARC stable cell lines, L5 and L24, were treated with 0-500 JIM H202for 8 h. Cell viability was assessed using the calcein-acetoxymethyl (AM) cell viability assaywhich utilized non-fluorecent dye that is converted to a green-fluorescent calcein after AMester 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 finalconcentration of 5 pi.M. Fluorescence intensity was read following one hour (h) of substrateincubation (excitation 485 nm, emission 527 nm) using the'PECAN GENios fluorescentplate reader (Tecan, San Jose, CA).23.7 H9c2 stable and transient transfectionStably transfected H9c2 cells with pcDNA3-Neo or pcDNA3-ARC were obtained fromDr. 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, whichexpress high levels of ARC. 12An adenoviral vector expressing ARC (Ad-ARC) and an adenoviral vector expressinggreen 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 tocell 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.67Chapter 223.8 H9c2 myocyte differentiationMuscle differentiation was induced by culturing cells to 100% confluency, removingmedia containing serum, washing cells twice with DPBS and replacing medium with DMEMcontaining 1% horse serum (HS). Media was changed daily for 5 days. Differentiation wasassessed by measuring expression of differentiation markers by Western blot and wasquantified 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 randomlychosen microscopic fields.2.3.9 F-actin and nuclear staining of H9c2 cellsH9c2 cells transfected with ARC or vector alone were grown on glass coverslips anddifferentiated as described in section 2.3.8. Cells were stained with AlexaFluor 488-labelledphalloidin and Hoechst 33342 (Molecular Probes, OR) to visualize F-actin and nuclei,respectively. Phalloidin is a water soluble bicyclic peptide derived from Amanita phalloidesmushrooms that binds strongly to F-actin. 13 Hoechst is a cell soluble, blue-fluorescentbisbenzimidazole derivative that binds to the minor groove of DNA. 14 Cells were washedtwice with DPBS, fixed with 2% paraformaldehyde for 10 min at room temperature andrewashed twice with DPBS. Cells were then permeabilized in a 0.1% Triton X solution for 5min and washed twice with DPBS. Cells were incubated with 1 pM AlexaFluor 488-labelledphalloidin and 1 pg/m1 Hoechst 33342 for 30 min, washed three times with DPBS beforeimaging. Slides were imaged using a Nikon Eclipse T6300 microscope and Spot digital68Chapter 2camera. The excitation and emission wavelengths for AlexaFluor 488 and Hoechst 33342 are350 nm and 461 nm, respectively.2.3.10 DEVDase activity assayDEVDase activity assays were performed to detect caspase-3/7-like activity, asdescribed previously. 15' 16 At day 0, 1, 3 and 5 post-differentiation, H9c2 cells transfectedwith ARC or vector alone were lysed in whole cell lysis buffer (1% NP-40, 20 mM Tris, pH8, 137 mM NaCI, 10% glycerol, 1 mM phenylmethyl sulfoxide, 0.15 U/ml aprotinin, and 1mM sodium orthovanadate). Lysates (0.3 mg/ml) or buffer as control were plated in triplicateand incubated at 37°C for 15 min. Acetyl-DEVD-7-amino-4-methylcoumarin (Ac-DEVD-AMC) (37.5 mg/ml, Calbiochem) caspase-3 substrate was added and relative florescenceunits (RFU) were measured after 2 h at 37°C using the 1 ECAN GENios fluorescence platereader (ex: 380 nm, em: 460 nm).2.3.11 Statistical analysisAll results are expressed as mean ± SE, and analyzed with GraphPad Prism 4 software usingone-way analysis of variance (ANOVA) with multiple comparisons performed by Students'T test. The results of statistical tests were considered statistically significant at p<0.05.69Chapter 22.4 Results2.4.1 Native ARC expression in endothelial and smooth muscle cell linesNative levels of ARC expression were assessed in cultured HUVECs and HCASMCsby 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 arehigher in HUVECs than in HCASMCs. A representative image of ARC transcript levels isshown in Figure 2.2(A). We then compared protein expression levels of ARC in these celllines. Again we were able to detect ARC expression in both lines with higher expressionlevels observed in HUVECs. A representative Western blot is shown in Figure 2.2(B).2.4.2 TAT-ARC purification and transduction in vascular cellsARC is a splice variant of the Nop30 protein. Although they have poor homology atthe protein level, as a result of a frame shift cause by an alternate splicing at exon 2, there isonly one unique sequence of 10 nucleotides contained in ARC that is not in the sequence ofNop30. This sequence is unfortunately a poor target for siRNA. Thus we decided to examinethe effect of increased ARC levels via TAT-fusion protein transduction. TAT-ARC andTAT-0-gal were expressed in BL21(DE3)pLysS bacteria, purified using Ni-NTA columns andwere then desalted. We routinely obtained purified protein concentrations greater that 2mg/ml. Texas-red conjugated TAT-ARC and TAT-0-gal were successfully transduced intoboth HCASMCs and HUVECs in culture (Figure 2.3). The transduced protein appears70Chapter 2AARC18SRT-PCRSMC ECBWestern blotARCSMC^ECFigure 2.2 HCASMCs and HUVECs express ARC.(A) Representative RT-PCR experiment (of n=3) showing detection of ARC transcripts incultured HCASMCs (SMC) and HUVECs (EC). (B) Representative Western blot (of n=3)demonstrating ARC protein expression.71Chapter 2HCASMC HUVECTAT-ARCUntreatedFigure 2.3 TAT-ARC fusion protein transduction into HCASMCs and HUVECs.Representative fluorescent images of HCASMCs and HUVECs were treated with Texas-redconjugated TAT-ARC fusion protein demonstrating successful protein uptake.72Chapter 2punctate (Figure 2.4(A)) and was taken up in a concentration-dependent manner (Figure2.4(B)).2.4.3 ARC over-expression does not protect against H202 treatment.ARC' s ability to protect against H 202 treatment was measured in both HUVECs andHCASMCs. Cells were pre-treated with TAT-ARC or TAT-13-gal (control) and were thenexposed to 0.6 mM H202 for 4 h. Viability was measured using the MTS assay (see Figures2.5 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 againstH202 treatment; however, high levels of transduction (>100nM) were required to see anyprotective effect and TAT-ARC treatment was no more protective than treatment with theTAT-13-gal fusion protein.2.4.4 Functional overexpression of ARC in pre-differentiated H9c2 cellsSince ARC has previously been shown to be protective in cardiomyocytes andskeletal muscle4' 5' 12.17 we turned to the rat embryonic myocyte cell line H9c2. H9c2 celllines stably overexpressing ARC or Neo (control) were created by selecting clones frompcDNA3 transfected cells. Two clones, L5 and L24, were selected due to their highexpression levels of ARC (Figure 2.7 (A)). Both clones were used in subsequent analyses inorder to reduce the possibility that the effects observed are resulting from a gene disruption73Chapter 2BTAT-ARC411.1111.1111110^ f3-actin0^62.5^125^250^500^1000TAT-ARC treatment (nM)Figure 2.4 TAT-ARC uptake into HUVECs and HCASMCs is punctate andconcentration-dependent.(A) High magnification fluorescent image of Texas-red conjugated TAT-ARC internalizationinto HUVECs showing punctuate protein distribution. (B) Western blot of ARC proteinfollowing TAT-ARC transduction into HUVECs showing concentration-dependent uptakeagainst [3-actin protein control.74Chapter 212010008000"6 60:acrs^40200 TAT-ARCTAT-13-gal0^50^100^250^500Concentration of TAT-fusion protein (nM)Figure 2.5 TAT-ARC does not confer greater protection against H20 2 in HUVECs thanTAT-13-gal controlHUVECs were pretreated with increasing concentrations of TAT-ARC and TAT-13-gal andsubjected to 0.6 mM H202 for 4 h. Data are shown as percent viability of untreated cells asmeasured by the MTS viability assay. Data represents the mean ± SD of 4 experiments, 3repeats/experiment.75Chapter 2 1401201008060402000C0U00 TAT-ARCE TAT-0-gal0^50^100^250^500Concentration of TAT-fusion protein (nM)Figure 2.6 TAT-ARC does not confer greater protection against H202 in HCASMCsthan TAT-13-gal controlHCASMCs were pretreated with increasing concentrations of TAT-ARC and TAT-13-gal andsubjected to 0.6 mM H202 for 4 h. Data are shown as percent viability of untreated cells asmeasured by the MTS viability assay. Data represents the mean ± SD of 4 experiments, 3repeats/experiment. Significance was calculated using a Student's t-test, p-values > 0.1.76Chapter 2as a consequence of vector integration into the host cell genome rather than from theoverexpression of ARC. The functionality of ARC was confirmed in these cells lines byexamining cell viability following exposure to hydrogen peroxide (Figure 2.7 (B)). H9c2-Neo control cells demonstrated concentration-dependent loss in viability after 8 h oftreatment with 11202 concentrations between 0 and 500 RM. The H9c2-ARC clones, L5 andL24, 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 onlythe highest concentration of 500 i.iM (73.9 ± 4.2% for L5, 82.5 ± 12.5% for L24, compared to41.1 ± 4.0% of Neo cells; expressed as mean ± SE).2.4.5 Characterization of H9c2-ARC cell differentiationWe then wanted to examine the role of ARC overexpression in differentiated H9c2cells. We induced myotube differentiation using standard protocols by reducing serumconcentration from 10% FCS to 1% HS. Cells were visualized at 0, 3 and 5 days after theinduction of differentiation. H9c2-Neo cells demonstrated myoblastelongation/differentiation; however, elongation was attenuated in H9c2-ARC cells (Figure2.8). Elevated myotube disarray and disorganization were observed at day 3 and 5 in theARC overexpressing cells compared to that of the H9c2-Neo cells. In addition to myotubeelongation, multi-nucleation was also observed in the H9c2-Neo at a rate of 11.2 ± 5.1%, butwas absent in the H9c2-ARC cells (Figure 2.9 and 2.10).To further assess the influence of ARC overexpression on myoblast differentiation, thestatus of muscle specific proteins myogenin and troponin T were evaluated. Myogenin andtroponin T were both highly expressed in differentiated (Day 3 and 5 post-differentiation)77140 _120 _100 _806040200H9c2/Neo^ H9c2/L5■ H9c2/L24BH9c2Neo^L5^L24- ARC- 13-actin1111 INAChapter 20^31.3^62.5^125.0^250.0^500.0H202 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 ARCcells were treated with various concentrations of H202 and cell viability was assessed usingthe calcein-AM cell viability assay at 8 h post-treatment. Values are expressed as the percentviability the test group compared to that of untreated H9c2-Neo cells. Bars represent themean ± SE (n = 3).78Chapter 2Day 0^Day 3^Day 5Figure 2.8 Overexpression of ARC prevents myogenic differentiation.Representative morphological changes of Neo control and ARC overexpressing stable celllines, L5 and L24, at day 0, 3 and 5 following the induction of differentiation. Characteristicalignment and elongation of cells is observed in control cells but is not apparent in ARCoverexpressing cell lines.79Chapter 2Figure 2.9 Overexpression of ARC prevents myogenic differentiation.Fluorescent staining of ARC overexpressing H9c2 cells at day 5 post-differentiation. H9c2cells transfected with ARC, or vector alone were stained with AlexaFluor 488-labelledphalloidin for F-actin (green). Cell nuclei were counterstained with Hoechst (blue). Arrowsindicate multi-nucleated cells.80Chapter 218 -16 -14 H12 -10 -86 -420Neo^ARCFigure 2.10 Overexpression of ARC prevents myogenic differentiation.Myogenic differentiation was quantified by the number of differentiated cells which showedat least three nuclei and expressed as a percentage of the total number of nuclei in tenrandomly chosen microscopic fields. The results shown are mean ± SD (n=10) andsignificance was determined by Student's t-test. (*=p<0.05).810Ca"12Chapter 2H9c2-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 weretransiently transduced with an adenovirus ARC construct, the muscle differentiation markerswere significantly reduced (Figure 2.12).2.4.6 ARC expression during differentiationWe then examined the native expression levels of ARC throughout H9c2 cell differentiation.ARC was undetectable in undifferentiated H9c2 cells. However, ARC expression rose todetectable levels by day 3 following differentiation and by day 5 ARC levels had reachedmaximal and sustained expression levels (Figure 2.13).2.4.7 Caspase-3 activation during H9c2 differentiationTo examine whether caspase-3 is activated during myotube differentiation, wemeasured caspase-3 activity by the cleavage of Ac-DEVD-AMC substrate. As shown inFigure 2.14, at day 0 post-differentiation, caspase-3 activity was not different between Neoand ARC overexpressing cells (Neo: 4400 ± 504 RFU, ARC: 4192 ± 2672 RFU; expressedas mean ± SD). However at day 1 post-differentiation caspase-3 activity significantlyincreased in the Neo cells, implying an important role of caspase activation in celldifferentiation. Overexpression of ARC prevented caspase-3 activation at day 1 post-differentiation (16108 ± 1135 RFU for ARC cells compared to 39736 ± 1796 RFU for Neocells). 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).82Chapter 2Day 0Neo L5 L24Day 3Neo L5 L24Day 5Neo L5 L24-Troponin T-Myogenin-ARC-13-actinFigure 2.11 ARC stable overexpression prevents the expression of the muscle-specificmarkers troponin T and myogenin.At day 0, 3 and 5 post-differentiation, cell lysates were collected and Western blotting wasperformed to determine troponin T, myogenin and ARC expression. The same membraneswere blotted with an antibody against 13-actin to illustrate equal protein loading. The data arerepresentative of three different experiments.83Chapter 2Day 0^Day 3^Day 5Ad- Ad- Ad- Ad- Ad- Ad-GFP ARC GFP ARC GFP ARC-Troponin T-Myogenin-ARC-I3-actinFigure 2.12 Transient ARC 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 wasperformed to determine troponin T, myogenin and ARC expression. The same membraneswere blotted with an antibody against 13-actin to illustrate equal protein loading. The data arerepresentative of three different experiments.84Chapter 2Neo0^3^5^7 Days post-differentiation-ARC-I3-actinswam airmii 4iggie Ma.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^ NeoII ARCM 4500LLCC 40003500 3000(,) 2500CCICI 2000LIU 15005000 =it1000 -Chapter 2P<0.005Day 0^Day 1^Day 3^Day 5Figure 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 wasdetermined. The data shown are mean ± SD (n=3) and significance was determined by Student's t-test.86Chapter 22.5 DiscussionARC expression was initially thought to be restricted to the highly differentiatedtissues of skeletal and cardiac muscle. 1 Since that time ARC has also been found to beexpressed in other cells types, most notably cancer cells. 18,19 Here we demonstrate that ARCis also expressed in endothelial cells and smooth muscle cells. Although ARC expression isusually associated with muscle phenotypes we observed greater levels of both ARC transcriptand protein expression in endothelial cells compared to smooth muscle. Interestingly, proteintransduction of ARC into these cells types did not confer greater protection than the controlprotein 13-gal to treatment with H202. It is unclear why TAT-ARC is not exerting a protectiveeffect in these cells. One possibility is that TAT-ARC is not being properly folded ortransported and thus is not biologically active in these cells. TAT-ARC has been shown to beprotective against oxidative injury in cardiac tissue s and ARC overexpression has beenshown 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 andcardiac muscle that its expression must be repressed in these tissues to allow differentiationto occur. Apoptosis is a critical physiological process that is essential for normal tissuedevelopment and homeostasis. Dysregulation of this form of cell death is associated withnumerous pathological conditions. Recent studies suggest that apoptosis and differentiationshare common pathways in muscle cells. 2°-22Actin fibre disassembly and reorganization are conserved features of both apoptosis andmyoblast differentiation. Similarly, caspase activity is a key component of both apoptosis and87Chapter 2skeletal muscle differentiation. 2° Caspase-3 inhibition reduces myotube/myofibre formationas well as expression of muscle-specific proteins during myogenesis. 2° Further, dysregulatedmyoblast differentiation and apoptosis in response to certain cytokines may be associatedwith increased muscle wasting in chronic disease states such as infection, Acquired ImmuneDeficiency Syndrome (AIDS) and cancer. 23 Interestingly, tumour necrosis factor alpha (TNF-a), a principle cytokine associated with cachexia, is involved in the regulation of skeletalmuscle differentiation and apoptosis.23 Thus, it is exciting to speculate that proteins whichinhibit both differentiation and apoptosis could attenuate such degenerative diseases bypreventing 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 normalconditions and are mono-nucleated. This cell line is also a well-characterized model ofdifferentiation. When H9c2 cells are exposed to reduced serum concentrations at confluence,they fuse and differentiate into elongated, multi-nucleated myotubes. 24 Although this cell lineexhibits some differences from primary cells, H9c2 cells share many of the properties ofprimary cardiomyoblasts and skeletal muscle 24-27 and can be easily propagated and stablytransfected, making them an ideal model for this study.ARC is highly expressed in terminally differentiated cardiac and skeletal muscle cells. 1However, we were unable to detect ARC expression in undifferentiated H9c2 rat myoblastcells leading us to ask questions regarding the regulation of ARC expression during myoblastdifferentiation and the importance of this regulation on the differentiation process.88Chapter 2ARC expression increased by day 3 following differentiation and reached maximal andsustained levels by day 5. To understand whether regulation of ARC expression duringdifferentiation is important to the process of differentiation itself, we examined the effect ofARC over-expression using pre-differentiated H9c2 cells. H9c2 cells overexpressing ARCwere unable to differentiate as indicated by morphological characteristics such as myotubeelongation and multinucleation.Myogenic differentiation, characterized by cell growth arrest, myoblast alignment,elongation, and fusion of mono-nucleated myoblasts into multi-nucleated myotubes, isdependent on the expression of the MyoD family of basic helix-loop-helix (bHLH)transcription factors, which includes MyoD, MyfS, myogenin, and MRF4. 28 Uponstimulation, myoblasts are induced to express muscle regulatory factors, which in turn leadsto the expression of muscle-specific genes. To further assess the influence of ARCoverexpression on myoblast differentiation, the status of muscle specific proteins myogeninand troponin T were evaluated. Myogenin and troponin T were both highly expressed indifferentiated (day 3 and 5 post-differentiation) H9c2-Neo cells but were minimallydetectable in the ARC-overexpressing, L5 and L24, H9c2 cell lines (Figure 2.11). Theseresults were confirmed using H9c2-Neo cells that were transiently transduced with anadenovirus ARC construct. Thus, inhibition of ARC expression is vital for the properdifferentiation of these cells. Whether premature ARC expression culminates in abnormalcardiovascular development is unclear and requires further investigation.Recent studies have suggested that caspase-3 activity is required for skeletal muscledifferentiation. 20 As ARC inhibits caspase-mediated apoptosis, we examined whethercaspase-3 is activated during myotube differentiation and whether overexpression of ARC89Chapter 2prevents that activation. Caspase-3 activity significantly increased at day 1 post-differentiation and ARC overexpression prevented caspase activation. As ARC does notinteract directly with caspase-3, it is likely modifying caspase-3 activation through itsinteractions with caspase-2, 8 or the Bcl-2 protein Bax, as has previously been shown. 3' 29These findings suggest that ARC expression must be attenuated or absent to allow for earlydifferentiation events including caspase-3 activation and that it is at this early stage ofdifferentiation during which ARC impacts the differentiation process. Our initial finding thatARC is endogenously expressed at undetectable levels until day 3 post-differentiation (i.e.after caspase-3 is activated) supports this hypothesis. Therefore, the inhibition ofdifferentiation markers, such as myogenin and troponin-T, in the ARC overexpressing celllines are likely a result of these upstream events. This hypothesis would explain whyendogenous expression of ARC at day 3 and day 5 does not prevent differentiation. Theinduction of ARC following caspase-3 activation would likely be beneficial as it may protectproperly differentiating cells from apoptosis.It is becoming increasingly more apparent that ARC is a multi-faceted anti-apoptoticprotein. Several mechanisms have been proposed as to how ARC attenuates apoptosisincluding: inhibition of caspases, mitochondrial membrane depolarization, DISC formation,Bax activation, and cytochrome c release or K+ currents. 1-5' 12 The results of this studydemonstrate a novel role for ARC in myoblast differentiation and suggest that ARCexpression is tightly controlled throughout the differentiation process in order to allow forinitiating events such as caspase-3 activity.The challenge for future studies will be to further delineate the detailed mechanisms bywhich ARCs expression is regulated and contributes to myocyte differentiation as well as the90Chapter 2potential role of ARC in myocyte disarray and disease. Reduced troponin T expression,which we have shown can be induced by ARC expression during differentiation, contributesto myocyte disarray3° and mutations in this gene are associated with myocyte disarray and15% of all cases of familial hypertrophic cardiomyopathy.31 Myofibrillar disarray is alsocommonly associated with familial hypertrophic cardiomyopathy, the leading cause ofsudden 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 againstoxidant injury induced by H20 2 are preliminary in nature. It is possible that ARC is able toprotect against oxidant stress induced through other pathways. Since we were able to detectARC expression in both endothelial and smooth muscle cell lines it seems likely that ARCdoes play a role in these cells. One possibility is that the native levels of ARC are highenough to confer maximal protection via its many pathways. Although this is possible, othercell types, such as cardiomyocytes, that express much higher levels of ARC do still gain anadditional protective effect upon TAT-ARC transduction. 5 Our ultimate research goal is toexamine the relationship between ischemic injury and CAV. Given the intense interest byother groups in examining the role of ARC in myocardial ischemic injury and our failure toobtain preliminary evidence demonstrating a protective role for ARC in vascular ischemicinjury we selected to examine other proteins. One such group of proteins are the cytochromep450 2C enzymes. The role of these enzymes in peri-transplant ischemic injury and CAV areexamined in the subsequent chapters of this thesis.91Chapter 22.6 Bibliography1. Koseki T, Inohara N, Chen S, et al. ARC, an inhibitor of apoptosis expressed inskeletal muscle and heart that interacts selectively with caspases. Proc Natl Acad SciUSA.  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 stress-mediated cell death by preserving mitochondrial function. J Biol Chem. Sep 72001;276(36):33915-33922.3. Nam YJ, Mani K, Ashton AW, et al. Inhibition of both the extrinsic and intrinsicdeath pathways through nonhomotypic death-fold interactions. Mol Cell. Sep 242004;15(6):901-912.4. Ekhterae D, Lin Z, Lundberg MS, et al. ARC inhibits cytochrome c release frommitochondria and protects against hypoxia-induced apoptosis in heart-derived H9c2cells. Circ Res. Dec 9 1999;85(12):e70-77.5. Gustafsson AB, Sayen MR, Williams SD, et al. TAT protein transduction intoisolated perfused hearts: TAT-apoptosis repressor with caspase recruitment domain iscardioprotective. Circulation. Aug 6 2002;106(6):735-739.6. Schwarze SR, Ho A, Vocero-Akbani A, et al. In vivo protein transduction: delivery ofa biologically active protein into the mouse. Science. Sep 3 1999;285(5433):1569-1572.7. Becker-Hapak M, McAllister SS, Dowdy SF. TAT-mediated protein transduction intomammalian cells. Methods. Jul 2001;24(3):247-256.8. Wadia JS, Dowdy SF. Protein transduction technology. Curr Opin Biotechnol. Feb2002;13(1):52-56.9. Wadia JS, Dowdy SF. Modulation of cellular function by TAT mediated transductionof 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 TATfusion 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 nativehistidine-tagged protein expressed by recombinant vaccinia virus. Proc Nall Acad SciUSA.  Oct 15 1991;88(20):8972-8976.12. Ekhterae D, Platoshyn 0, Zhang S, et al. Apoptosis repressor with caspase domaininhibits 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;15:285-300.14. Portugal J, Waring MJ. Assignment of DNA binding sites for 4',6-diamidine-2-phenylindole 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 apoptosis-inducing factor and cytochrome c during smooth muscle cell apoptosis. Am J Pathol.Jul 2001;159(1):305-311.92Chapter 216. Granville DJ, Shaw JR, Leong S, et al. Release of cytochrome c, Bax migration, Bidcleavage, and activation of caspases 2, 3, 6, 7, 8, and 9 during endothelial cellapoptosis. Am J Pathol. Oct 1999;155(4):1021-1025.17. Gustafsson AB, Gottlieb RA, Granville DJ. TAT-mediated protein transduction:delivering biologically active proteins to the heart. Methods Mol Med. 2005;112:81-90.18. Mercier I, Vuolo M, Madan R, et al. ARC, an apoptosis suppressor limited toterminally differentiated cells, is induced in human breast cancer and confers chemo-and radiation-resistance. Cell Death Differ. Jun 2005;12(6):682-686.19. Wang M, Qanungo S, Crow MT, et al. Apoptosis repressor with caspase recruitmentdomain (ARC) is expressed in cancer cells and localizes to nuclei. FEBS Lett. Apr 252005;579(11):2411-2415.20. Fernando P, Kelly JF, Balazsi K, et al. Caspase 3 activity is required for skeletalmuscle differentiation. Proc Natl Acad Sci U S A. Aug 20 2002;99(17):11025-11030.21. Kageyama K, Ihara Y, Goto S, et al. Overexpression of calreticulin modulates proteinkinase B/Akt signaling to promote apoptosis during cardiac differentiation ofcardiomyoblast H9c2 cells. J Biol Chem. May 31 2002;277(22):19255-19264.22. van den Eijnde SM, van den Hoff MJ, Reutelingsperger CP, et al. Transientexpression of phosphatidylserine at cell-cell contact areas is required for myotubeformation. J Cell Sci. Oct 2001;114(Pt 20):3631-3642.23. Coletti D, Yang E, Marazzi G, et al. TNFalpha inhibits skeletal myogenesis through aPW1-dependent pathway by recruitment of caspase pathways. Embo J. Feb 152002;21(4):631 -642.24. Kimes BW, Brandt BL. Properties of a clonal muscle cell line from rat heart. Exp CellRes. Mar 15 1976;98(2):367-381.25. Hescheler J, Meyer R, Plant S, et al. Morphological, biochemical, andelectrophysiological characterization of a clonal cell (H9c2) line from rat heart. CircRes. Dec 1991;69(6):1476-1486.26. Kolodziejczyk SM, Walsh GS, Balazsi K, et al. Activation of JNK1 contributes todystrophic muscle pathogenesis. Curr Biol. Aug 21 2001;11(16):1278-1282.27. Pagano M, Naviglio S, Spina A, et al. Differentiation of H9c2 cardiomyoblasts: Therole of adenylate cyclase system. J Cell Physiol. Mar 2004;198(3):408-416.28. Buckingham M. Molecular biology of muscle development. Cell. Jul 151994;78(1):15-21.29. Gustafsson AB, Tsai JG, Logue SE, et al. Apoptosis repressor with caspaserecruitment domain protects against cell death by interfering with Bax activation. JBiol Chem. May 14 2004;279(20):21233-21238.30. Peschiaroli A, Figliola R, Coltella L, et al. MyoD induces apoptosis in the absence ofRB function through a p21(WAF1)-dependent re-localization of cyclin/cdkcomplexes to the nucleus. Oncogene. Nov 21 2002;21(53):8114-8127.31. Sehnert AJ, Huq A, Weinstein BM, et al. Cardiac troponin T is essential in sarcomereassembly and cardiac contractility. Nat Genet. May 2002;31(1):106-110.93Chapter 3: Cytochrome p450 2C Contributes toPost-Ischemic Vascular Dysfunction23.1 IntroductionCardiac I/R contribute to MI and is also a major complication in surgical treatmentssuch as cardiac transplantation, balloon angioplasty and coronary bypass. I/R is associatedwith a burst of ROS which is thought to contribute to both vascular dysfunction andmyocardial damage.CYP enzymes, reviewed in section 1.4, are membrane-bound heme-containingterminal oxidases responsible for the oxidation, peroxidation and/or reduction of a largenumber of substances including cardiovascular drugs. Although the majority of CYP arefound in hepatic tissues, these enzymes are also expressed in extra-hepatic tissues such as theintestine, kidney, lung, heart, and blood vessels. CYP enzymes contribute to the cellulargeneration of ROS. 1 Superoxide anions (02'), hydrogen peroxide and hydroxyl radicals areproduced during the CYP reaction cycle when electrons for the reduction of the central hemeiron are transferred to the activated bound oxygen molecule.' Recent evidence indicates animportant role for CYP in the pathogenesis of several cardiovascular diseases. 2-8 We recentlyreported 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 ratLangendorff perfusion model of global ischemia as well as in a rabbit coronary ligation942 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 3model of focal ischemia. In the latter study, CYP inhibition also reduced post-ischemic CKrelease and superoxide generation while restoring coronary flow. 3 In a clinical setting,sulfaphenazole (SP) restored endothelium-dependent vasodilator responses in patients withmanifest coronary artery disease.2 However, the role of CYP 2C9 in I/R-mediated vasculardysfunction has not been examined.SP, a highly specific inhibitor of CYP 2C9 in humans, exerts its inhibitory effect bybinding to the heme group of CYP 2C9. Its specificity is mediated through interactionsbetween SP's phenyl substituent and the phenyl group of Phe 114 on CYP 2C9. 9 SP alsoinhibits CYP 2C6 in rats.10 SP is dose dependently specific for CYP 2C9 (humans) and CYP2C6 (rats).3.2 AimAs CYP 2C6/9 inhibition increases endothelium-dependent vasodilation in patientswith coronary artery disease and increases post-ischemic coronary flow we hypothesizedthat 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 endothelium-dependent, NO-mediated vasodilation in post-ischemic vascular dysfunction. Wedemonstrate for the first time that SP restores endothelium-dependent vascular functionfollowing I/R.95Chapter 33.3 Materials and Methods3.3.1 Heart perfusion and vessel cannulationExperimental protocols were approved by the Animal Care Committee of theUniversity of British Columbia. A copy of the animal care certificate is provided inAppendix I. Male Sprague-Dawley rats (300-350 g, n=10) were injected with sodiumpentobarbital (60 mg/kg) and heparin sulphate (1000 U/kg) intraperitoneally. After loss ofreflexes 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, MgSO47H2O1.17, CaC12 1.6, and glucose 11.1). Hearts were perfused in the Langendorff mode aspreviously described. 3 Hearts were randomly divided into three groups: i) I/R: hearts wereperfused 20 min followed by 30 min no-flow global ischemia and 15 min reperfusion withmodified Krebs' buffer, ii) I/R with SP (I/R+SP): hearts prepared as in the li/R group but withthe addition of SP (10 p.M) in the perfusate, and iii) control: hearts were perfused for the totalperfusion 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 theLangendorff apparatus and placed in a dissection dish with ice-cold buffer. Septal coronaryarteries (intraluminal diameter is between 190-290 tun at 20 mm Hg) were dissected andtransferred to the chamber of a pressure myograph. A Video Dimension Analyzer (LivingSystems Instrumentation, Burlington, VT, USA) was used to measure inner diameter asdescribed elsewhere.1196Chapter 33.3.2 Vasomotor responses in septal arteriesSeptal coronary arteries were pretreated with U46619 (1 tiM, Cayman Chemical, AnnArbor, MI) at 20 mm Hg. After a sustained constriction, tissues were exposed to ACh (1 nM-10 uM) added to the external reservoir, and final maintained diameters were recorded. Anidentical protocol was used to study the vasodilator effects of sodium nitroprusside (SNP, 1nM-10 uM), and isoproterenol (1 nM-10 uM). In addition, the constrictor responses tovarious concentrations of KC1 (20, 35, 66, 84 mM) were examined. At the end of eachexperiment, Krebs' buffer was substituted with Krebs' buffer containing no CaC12 and 2.0mM EGTA to achieve zero calcium and the maximal passive diameters.3.3.3 Dihydroethidium (DHE) staining of coronary blood vesselsHearts were flash frozen in liquid nitrogen following Langendorff perfusion, asdescribed above, and stained for superoxide production modified from methods previouslydescribed (Miller et al., 1998). Frozen hearts were sectioned at 20 ptm on a ThermoShandoncryostat. DHE (Molecular Probes, Eugene, OR) was prepared under N2 gas by dissolving to 1mg/m1 in DMSO and then dilution in phosphate buffered saline (PBS). Sections were treatedin 2 tiM DHE for 30 min at 37°C under N2 gas. Sections were washed twice with PBS, coverslips were applied and slides and were imaged immediately. Imaging was performed on anEclipse TE300 fluorescent microscope (Nikon; excitation: 488 nm, emission: 610 nm) underidentical 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 ofthe arteries from the untreated hearts representing 100%.97Chapter 33.3.4 Measurements of dityrosine in coronary effluentsDityrosine measurements were performed utilizing the method developed by Yasminet a/. 12 L-tyrosine (0.3 mM) was added to Kreb's buffer and Langendorff perfusions werecarried out as described above. Coronary effluent fractions were collected. Upon reactionwith peroxynitrite, L-tyrosine is converted to dityrosine which absorbs at 320 nm.3.3.5 Statistical analysisAll results are expressed as mean ± SE, and analyzed with NCSS 2000 and PASS2000 software using one-way analysis of variance (ANOVA) and/or repeated-measuresANOVA with multiple comparisons performed by Bonferroni's test. -LogEC50 (pD2) wascalculated by Graphpad Prism®, version 3.02. The results of statistical tests were consideredstatistically significant at p<0.05.3.4 Results3.4.1 Endothelium-dependent vasomotor responsesTonic contractions for U46619 (1 JAM), a stable analog of thromboxane A2, were notaltered 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 shows98174A 2905 min WashU46619a)250141E-4 227.4-4)1-1134ControlI/R+ 10 [tM SPI/RBO ControlO I/R I/R+10 pM SPChapter 3Log [ACh] MFigure 3.1. Sulfaphenazole (SP) restores post-ischemic endothelium-dependent NO-mediated vasodilation.(A) Representative traces showing SP attenuates impaired endothelium-dependentvasorelaxation to ACh. (B) Concentration-response curves to ACh, showing that SPincreased sensitivity and maximal responses to ACh (n=5, *p<0.05).99Chapter 3representative traces (A) and response curves (B) demonstrating a rightward shift in theacetylcholine (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 (10pM) with a pD2 of 7.3±0.1 (n=5, p<0.005, I/R+SP vs. I/R).3.4.2 Endothelium-independent vasomotor responsesVasodilator responses to SNP were also reduced after I/R (Figure 3.2A, B). SP wasnot 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, controlvs. UR and I/R+SP). Likewise, SP (10 JIM) failed to restore I/R-induced impairment ofisoproterenol-mediated vasodilation (Figure 3.3A, B). The maximal responses elicited byisoproterenol 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 KCldemonstrated 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 productionDHE 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 the100 151 _U46619Chapter 3A 2811$. ,..HW 2334.4cts"t:J 243731 g 161ControlI/R+ 1011M SPUR5 min^SNP (1 nM — 10µM)^WashB^1008060COx 40wccs) 20r.   ^cl Control0 I/R I/R+10 uM SP"■ -7Log[SNP] MFigure 3.2. SP does not restore post-ischemic endothelium-independent vasodilationproduced 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 endothelium-independent vasodilation.101^ ControlO I/R• I/R+1 0 pM SPChapter 3AB217I1120Vg 90505301-11695 min10080604020ControlI/R+10 tiM SPURU4661 9Isoproterenol^Wash(1 nM — 10 ptM)-9^-8^-7^-6Log [Isopoterenol] MFigure 3.3 SP does not restore post-ischemic endothelium-independent vasodilationproduced 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 improveendothelium-independent vasodilation.102--c"— Control— 0-- I/R- 1113+10 pM SPChapter 380 -O 60 -:a70 40OC.)o4! 20 -20^40^60^80^100[KCI] MFigure 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 noadded effect (n=5, p>0.95).00-20103Chapter 3PBS Control^DihydroethidiumUntreated-10 pM SPFigure 3.5 SP reduces ROS production following I/R.Representative traces demonstrating that SP reduces relative fluorescent intensity ofdihydroethidium staining following I/R.104*I^Chapter 3120100806040200Untreated^10µM SPFigure 3.6 SP reduces ROS production following UR.Mean ± SE of the fluorescent intensity of dihydroethidium staining following UR in arterialwalls (control n=6, SP n=5, * p<0.005).10515105Time (min)E0.040.03C)0.02vii 0.01Chapter 3Figure 3.7 Peroxynitrite measurements in post-ischemic coronary effluent.Dityrosine conversion from L-tyrosine as measured by absorbance at 320 nm. L-tyrosine wasadded to the perfusate during Langendorff-perfusion induced I/R.106Chapter 3relative 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 DiscussionIncreased intracellular calcium levels following I/R induces the activation ofphospholipase A2 (PLA2) and the subsequent hydrolysis of AA from membranephospholipids. 13-16 AA is metabolized by CYP 2C9 into EETs, however, superoxide is alsogenerated during this reaction cycle.'' Superoxide readily reacts with NO  to produceONOO- and it has been proposed that NO , scavenging due to CYP 2C9-mediated superoxideproduction leads a reduction of NO  bioavailability that impairs endothelium-dependentvascular 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 thatSP restores endothelium-dependent, NO-mediated vasodilation in patients with coronaryartery disease2 in combination with our recent findings that SP significantly reduces infarctsize caused by I/R3, we hypothesized that SP attenuates post-ischemic endothelialdysfunction.Although the rat equivalent of CYP 2C9 has not been fully characterized, a CYP2C9-like isozyme that shares immunoreactivity and is selectively inhibited by SP has beendetected in rat arteries. 3' 20 CYP 2C6 is a putative rat homologue for human CYP 2C9. Insupport of this, SP has been shown to inhibit rat CYP 2C6 but not other members of the ratCYP 2C family. 1°107Chapter 3Here 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 post-ischemic impairment of endothelium-independent vasodilation (SNP, isoproterenol).Fichtlsherer et al. 2 also observed a similar trend where SP had no effect on impairedresponses to SNP in patients with coronary artery disease versus normal controls. Impairmentof endothelium-independent vasodilation was not due to a loss in smooth muscle cellcontractility as responses to KCl were similar in all three treatment groups. These resultsindicate that SP is acting through an endothelium-specific mechanism. SP's inability torestore responses to either SNP or isoproterenol, indicate that it is not acting by alteringguanylate 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 areduction in superoxide formation and consequent increase in NO  bioavailability. Figure 3.8shows a diagram of the proposed mechanism. Chloramphenicol, a potent of inhibitor of CYP2C6/9, 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 wallROS production. To examine this question, superoxide formation was assessed by stainingwith DHE following FR with or without SP assessed. DHE is converted to ethidium in thepresence of ROS and is most highly reactive with superoxide. Consequent ethidium stainingis visible by fluorescent microscopy. DHE conversion was quantified in the area aroundvascular walls. There was a significant reduction (-60%) of superoxide in vessels of heartspretreated with SP. These results indicate that CYP 2C9 contributes to FR-induced vascular108I/R Injury0,01111111181.101011040111110M141111$0111100001111111$1111111101111011101111101001011W1111110111011140110$1110114114114trifi[Ca2i]c ---.Phospholipase A2Arachidonic acidON00-EET and 20-HETE11$1:14111141111011111*111001110011101111144111:01$11101111111:14:01:14011411:101:14$111e1.401011111111111021*:41)1011101101$11111$1110111011111.111,41.1101111,ItItititiglitititilltItatitit10141104111;111111111111111011$101111111$111111#11111111011110111101411141$111;1411:1101n$14:4101110113$01141$11111$101111SMCVasodilationChapter 3Figure 3.8 Proposed mechanism of CYP 2C induced impaired post-ischemicvasodilation.Upon I/R, [Ca21, increases activating phospholipase A2 hydrolysis of arachidonic acid fromphospholipid membranes. AA is metabolized by CYP 2C leading to the production of EETsand 20-HETE and 02-. . 02 then reacts with NO  forming ONOO- and reducing thebioavailability of NO  and its ability to induce vasodilation. ONOO- can also inhibitvasodilation.109Chapter 3superoxide production and supports the hypothesis that CYP 2C9 mediates post-ischemicvascular dysfunction by reducing NO  bioavailability.SP has previously been shown to have no effect on complex I, II, and IV ofmitochondrial respiration and does not reduce superoxide generated via NADPH oxidase orxanthine oxidase. 1'3 Therefore, SP is not acting as a general antioxidant and is likely actingthrough specific inhibition of CYP 2C6/9; a known producer of superoxide. Inhibition ofROS production following UR has been examined using several experimental models thatemploy superoxide dismutase and antioxidants. Although these studies have demonstratedthe role of ROS in I/R they have not resulted in the development of effective treatments toalleviate I/R injury. SP presents a promising alternative strategy at reducing ischemic injuryas we have shown that it significantly decreases superoxide production and improvesvascular function. Treatment with SP is particularly promising in the context of cardiacsurgical procedures such as cardiac transplantation, balloon angioplasty and coronary bypasswhere 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 bothendothelium-dependent and independent vasodilation, ii) SP selectively restores post-ischemic endothelium-dependent, NO-mediated vasodilation and iii) SP reduces UR-inducedsuperoxide production. Our study indicates that SP confers a protective effect in post-ischemic vascular dysfunction through a reduction of CYP 2C6/9-mediated superoxideproduction. Thus, CYP 2C9 is a potentially important therapeutic target for patients withischemic heart disease and those undergoing surgical procedure where UR-injury is a factor.110Chapter 33.6 Bibliography1. Fleming I, Michaelis UR, Bredenkotter D, et al. Endothelium-derived hyperpolarizingfactor synthase (Cytochrome P450 2C9) is a functionally significant source ofreactive oxygen species in coronary arteries. Circ Res. Jan 19 2001;88(1):44-51.2. Fichtlscherer S, Dimmeler S, Breuer S, et al. Inhibition of cytochrome P450 2C9improves endothelium-dependent, nitric oxide-mediated vasodilatation in patientswith coronary artery disease. Circulation. Jan 20 2004 ;109(2): 178-183.3. Granville DJ, Tashakkor B, Takeuchi C, et al. Reduction of ischemia and reperfusion-induced myocardial damage by cytochrome P450 inhibitors. Proc Natl Acad Sci U SA. Feb 3 2004;101(5):1321-1326.4. Gross ER, Nithipatikom K, Hsu AK, et al. Cytochrome P450 omega-hydroxylaseinhibition reduces infarct size during reperfusion via the sarcolemmal KATP channel.J Mol Cell Cardiol. Dec 2004 ;37(6): 1245-1249.5. Hunter AL, Cruz RP, Cheyne BM, et al. Cytochrome p450 enzymes andcardiovascular disease. Can J Physiol Pharmacol. Dec 2004;82(12):1053-1060.6. Wang XL, Greco M, Sim AS, et al. Effect of CYP1A1 MspI polymorphism oncigarette smoking related coronary artery disease and diabetes. Atherosclerosis. Jun2002; 162(2):391-397.7. Wu S, Chen W, Murphy E, et al. Molecular cloning, expression, and functionalsignificance of a cytochrome P450 highly expressed in rat heart myocytes. J BiolChem. May 9 1997;272(19):12551-12559.8. Yasar U, Bennet AM, Eliasson E, et al. Allelic variants of cytochromes P450 2Cmodify the risk for acute myocardial infarction. Pharmacogenetics. Dec2003; 13(12):715-720.9. Melet A, Assrir N, Jean P, et al. Substrate selectivity of human cytochrome P4502C9: importance of residues 476, 365, and 114 in recognition of diclofenac andsulfaphenazole and in mechanism-based inactivation by tienilic acid. Arch BiochemBiophys. Jan 1 2003;409(1):80-91.10. Kobayashi K, Urashima K, Shimada N, et al. Selectivities of human cytochrome P450inhibitors toward rat P450 isoforms: study with cDNA-expressed systems of the rat.Drug Metab Dispos. Jul 2003;31(7):833-836.11. Skarsgard PL, Wang X, McDonald P, et al. Profound inhibition of myogenic tone inrat cardiac allografts is due to eNOS- and iNOS-based nitric oxide and an intrinsicdefect in vascular smooth muscle contraction. Circulation. Mar 212000;101(11):1303-1310.12. Yasmin W, Strynadka KD, Schulz R. Generation of peroxynitrite contributes toischemia-reperfusion injury in isolated rat hearts. Cardiovasc Res. Feb1997;33(2):422-432.13. Freyss-Beguin M, Millanvoye-van Brussel E, Duval D. Effect of oxygen deprivationon metabolism of arachidonic acid by cultures of rat heart cells. Am J Physiol. Aug1989;257(2 Pt 2):H444-451.14. Leong LL, Sturm MJ, Ismail Y, et al. Plasma phospholipase A2 activity in clinicalacute myocardial infarction. Clin Exp Pharmacol Physiol. Feb 1992 ;19(2): 113-118.111Chapter 315. Van der Vusse GJ, Reneman RS, van Bilsen M. Accumulation of arachidonic acid inischemic/reperfused cardiac tissue: possible causes and consequences. ProstaglandinsLeukot Essent Fatty Acids. Jul 1997;57(1):85-93.16. Williams SD, Gottlieb RA. Inhibition of mitochondrial calcium-independentphospholipase A2 (iPLA2) attenuates mitochondrial phospholipid loss and iscardioprotective. Biochem J. Feb 15 2002;362(Pt 1):23-32.17. Fulton D, McGiff JC, Wolin MS, et al. Evidence against a cytochrome P450-derivedreactive oxygen species as the mediator of the nitric oxide-independent vasodilatoreffect of bradykinin in the perfused heart of the rat. J Phannacol Exp Ther. Feb1997;280(2):702-709.18. Ogata K, Jin MB, Taniguchi M, et al. Attenuation of ischemia and reperfusion injuryof canine livers by inhibition of type II phospholipase A2 with LY329722.Transplantation. Apr 27 2001;71(8):1040-1046.19. Sargent CA, Vesterqvist 0, McCullough JR, et al. Effect of the phospholipase A2inhibitors quinacrine and 7,7-dimethyleicosadienoic acid in isolated globally ischemicrat hearts. J Pharmacol Exp Ther. Sep 1992;262(3):1161-1167.20. Earley S, Pastuszyn A, Walker BR. Cytochrome p-450 epoxygenase productscontribute to attenuated vasoconstriction after chronic hypoxia. Am J Physiol HeartCirc Physiol. Jul 2003;285(1):H127-136.112Chapter 4: Cytochrome p450 2C Contributes toCardiac Allograft Vasculopathy34.1 IntroductionMore than 3,000 heart transplants are performed worldwide annually. Currentimmunosuppressive regimens are very effective in preventing acute rejection. Unfortunately,chronic rejection associated with CAV remains a major hurdle to long-term graft survival ofall vascularized organ transplants. CAV is an accelerated and diffuse form of arteriosclerosisthat can be detected in up to 75% of heart transplant recipients following the first year oftransplantation." Although immunological mechanisms clearly play an important role in thepathogenesis of CAV, non-immunological mechanisms, such as peri-transplant UR injury,also contribute via direct damage or indirectly through cross-talk with immune responsesassociated with this type of vasculopathy. 3' 4The 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 evidencesupports a molecular and cellular basis for a causal relationship between UR injury duringtransplantation and the onset and progression of CAV. 6' 7 UR injury to endothelial cells mayprovide the initial trigger for atherogenesis by stimulating platelet adhesion, release ofgrowth factors, upregulation of MHC Class I and II expression, release of donor antigens,expression of adhesion molecules, and proliferation of vascular smooth muscle cells. (Reviewed1133 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 enzymescontribute peri-transplant ischemic injury and cardiac allograft vasculopathy. Am JTransplant.Chapter 41n3-91 Thus, attenuation of I/R injury would be of great benefit to transplant recipients not onlythrough the inhibition of direct cellular injury, but also indirectly through the aforementionedfactors that influence the allo-immune response. Several experimental models usingsuperoxide dismutase and antioxidants have demonstrated the importance of ROS in thepathophysiology of I/R injury 1"5; however, the development of effective treatments toalleviate reperfusion injury remains elusive.CYPs, as described in section 1.4, are membrane-bound heme-containing terminaloxidases that exist in a multi-enzyme system that includes a FAD/FMN-containing NADPHcytochrome p450 reductase and cytochrome b5. The CYP superfamily is responsible for theoxidation, peroxidation and/or reduction of vitamins, steroids, cholesterol, xenobiotics andthe majority of cardiovascular drugs in an oxygen and NADPH-dependent manner. Althoughthe vast majority of CYP are found in hepatic tissues, other CYP have been shown in recentyears 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 numberof cardiovascular diseases. 16-24 Previously, we discovered that the rat equivalent of CYP 2C9makes a significant contribution to superoxide generation and cell death associated with I/R  21injury. Recently, we demonstrated that the CYP 2C inhibitor sulfaphenazole (SP) increasesendothelium dependent vasodilation and decreases vascular superoxide production followingischemia and reperfusion. 25114Chapter 44.2 AimCYP 2C enzymes contribute to post-ischemic vascular dysfunction and cell death. Asischemic injury is thought to contribute to CAV, we hypothesized that CYP 2C willcontribute to the development of CAV. The aim of this study was to investigate whetherCYP 2C inhibition during the peri-transplant period would reduce post-transplant oxidativedamage and vascular remodelling associated with chronic cardiac rejection.4.3 Materials and Methods43.1 Heterotopic heart transplantationAll protocols were designed in accordance with the guidelines, and approved by, theanimal care committee of the University of British Columbia. A copy of the animal carecertificate for heterotopic heart transplantation is provided in Appendix I. Minorhistocompatibility antigen-mismatched rat heterotopic heart transplants were performedbetween 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 cardiactransplantation was performed as previously described. 1 Donors and recipients were treatedwith 5 mg/kg SP intraperitoneally (IP, Clinalpha, Laufelfingen, Switzerland), or vehiclecontrol 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 withheparinized saline and clamped distally. The right and left superior vena cavae (SVC) werethen ligated. The ascending aortas were cut below the brachiocephalic artery and the main115Chapter 4pulmonary arteries were cut proximal to their bifurcations. They were flushed withheparinized saline. Pulmonary veins were ligated together and the donor hearts were gentlydetached and placed in ice-cold heparinized saline. Recipients were anaesthetized withisofluorane (4% induction, 2% maintenance) Anastomoses were performed between theascending aortas of donor hearts and the abdominal aortas and between the pulmonaryarteries and the inferior vena cavae of the recipient animals. Buprenophrine was administeredsubcutaneously at 0.01 mg/kg immediately following surgery. A copy of the standardoperating procedure outlining the detailed protocol for this operation is provided in AppendixII.4.3.2 Tissue collectionAt 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) andxylazine hydrochloride (10 mg/kg). Thoracic and abdominal cavities were opened andtransplanted hearts were assessed for heartbeat. The circulatory system was flushed byinjecting 25 ml of Ringer's buffer at 80 mmHg into the right ventricle and cutting a smallincision in the right atria to allow fluids to drain. Rats were then perfusion fixed by replacingRinger's with 4% formalin 80 mmHg and allowing it to circulate as above. The native andtransplanted hearts were then removed rapidly, and transverse sections immersion fixed in10% formalin for 24 h before being embedded in paraffin.116Chapter 44.3.3 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 onformalin-fixed, paraffin embedded ventricular transverse sections. Briefly, sections weredeparaffinized by baking in a 60°C oven for 1 h followed by serial rehydration by immersionin 100% xylene (3X 5 min), 100% ethanol (2X 5 min), 90% ethanol (3 min), 70% ethanol (3min) and Tris-buffered saline (TBS, pH 7.4, 2X 5 min). Antigen retrieval was performed byboiling sections in citrate-citric acid buffer (pH 6.0) for 15 min, allowing sections to reachroom temperature and washing 2X with TBS. Exogenous phosphates were quenched byincubation in 10% H202 in TBS for 10 min and washing 2x with TBS. Sections were blockedby incubation of sections with blocking buffer (10% normal serum of the species thesecondary antibody was raised in) for 30 min. Sections were incubated in primary antibodiesovernight in blocking buffer at 4°C. Primary antibodies utilized were: 1:50 monoclonal a-ratKi-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 polyclonala-Von Willebrand Factor (Abcam Inc, Cambridge, MA). Sections were then washed 3X inTBS + 0.01% Tween 20 (TBST). Secondary detection was performed for 1 hr at roomtemperature with 1:350 biotinylated anti-secondary antibodies (Vector Laboratories,Burlingame, CA) in blocking buffer supplemented with 3% normal rat serum. The additionof 3% normal rat serum was required to reduce cross reactivity with rat IgG. Staining wasvisualized using the ABC kit (Vector Laboratories) followed by detection with thechromagens Nova Red or diaminobenzidine tetrahydrochloride (DAB, Vector Laboratories,117Chapter 4Burlingame, CA), and nuclei were counterstained with hematoxylin. Slides werecoverslipped using Aqua-mount® aqueous mountant (Lerner Laboratories, Pittburgh, PA).4.3.4 Histological assessment and quantificationFour 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 ratsper group, n=7 arteries/rat) for general, focal, sub-epicardial, sub-endocardial, andperivascular immune infiltration. Luminal narrowing in all visible medium to large sizecoronary arteries (30 days post-transplantation, n = 5 rats per group, 3 sections per rat, 7arteries per section) was evaluated on Movat's pentachrome stained sections. Briefly, Image-Pro PlusTM (MediaCybernetics, Silver Spring, MD) was used to quantify intimal and luminalareas, and percent luminal narrowing was calculated as the area of the lumen as a percentageof the combined area of the lumen and the intima. For assessments of immune infiltration thevascular wall area was defined as the region from the lumen to the outside of the medialsmooth muscle layer and the perivascular space (PVS) was defined as the region between themedial smooth muscle layer and the myocardium. These regions were traced and quantifiedusing Image-Pro P1usTM.4.3.5 Luminex analysisBlood was collected in accordance with the guidelines, and approved by, the animalcare committee of the University of British Columbia. A copy of the animal care certificate118Chapter 4for blood collection is provided in Appendix I. Tail vein blood was collected into heparinizedtubes 1 day prior to transplant and at days 1, 3, 5 and 7 post-transplant. Serum was isolatedby allowing blood to clot for 30 min and centrifuging for 15 min at 1000 g. Samples wereanalyzed using the rat cytokine/chemokine premixed LINCOplex 14-plex premix bead kit asper manufacturer's recommendations (LINCO Research, St. Charles, MO). Briefly, serumsamples were diluted 1:5 in LINCO Serum MatrixTM and standards were diluted in four-timesserial dilutions to 1:4096. The assay filter plate was blocked using LINCO Assay buffer for10 min at room temperature and was fluid was removed by gentle vacuum filtration. Dilutedsamples, standards and controls were incubated with premixed cytokine/chemokine detectionbeads overnight with agitation at 4°C at which time samples were drained by gentle vacuumfiltration and washed two times with LINCO washing buffer. Plates were developed by theaddition of the detection antibody cocktail and the streptavidin-phycoerythrin detectionsolution. Upon gentle vacuum filtration, plates were washed two times with wash buffer andbead-antibody complexes were solubilized in sheath fluid for analysis. Plates were analyzedon the Luminex FlowMetrix System (Qiagen, Mississauga, ON). Sample parameters requireda minimum of 50 events per bead. Samples were run in duplicate and compared to an 8-pointstandard curve developed using a 5-parameter logistic fit graph.4.3.6 8-Isoprostane measurementsFree 8-isoprostane measurements were performed using the 8-isoprostane EIA Kit onleft ventricular blood samples collected at sacrifice on day 4 and 7 post transplantation as permanufacturer's recommendations (Cayman Chemical Company, Ann Arbor, MI). Briefly,119Chapter 4samples were collected into tubes containing EDTA and 0.005% BHT (butylatedhydroxytoluene) 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 8-isoprostane affinity sorbent purification kit (Cayman). An equal volume of 15% KOH wasadded and samples were incubated at 40°C for 60 min. Four volumes of ethanol containing0.01% BHT was added and samples were vortexed, incubated for 5 min on ice andcentrifuged for 10 min at 1500 g. Supernatants were collected and ethanol was evaporatedunder nitrogen to less than 10% vol/vol. Purification required neutralizing the samples to pH7.4 by addition of 2 volumes of 1 M KH2PO4 and 1 volume of eicosanoid affinity columnbuffer. Samples were then added to pre-equilibrated 8-isoprostane affinity sorbent andallowed to bind for 60 min at room temperature, centrifuged briefly at 1500 g andsupernatant was discarded. Sorbent was then washed twice with ultrapure water and 8-isoprostanes were retrieved by incubating sorbent in ethanol Elution Solution with vortexing.Samples were dehydrated using a speed vacuum system and resuspended in EIA buffer forELISA. 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. EIAplates were developed by the addition of Ellman's reagent and 8-isoprostane tracer followedby incubation for 90 min at room temperature. Samples were measured via absorbancereadings at 412 nm on the Tecan GENios Rainbow absorbance plate reader (Tecan, San Jose,CA).120Chapter 44.3.7 Statistical analysisRepeated measures general linear model analysis was performed for Luminex resultsusing SPSS Statistical Software (Chicago, IL). Wilks' Lambda multivariate tests wereutilized to assess significance. For other assays, statistical differences between two groupswere determined using a Student's t-test. For both tests, a p-value (alpha error) of 0.05 or lesswas considered significant.4.4 ResultsThe heterotopic heart transplant model was utilized to assess the contribution of CYP2C to peri-transplant I/R injury and the development CAV. Donor hearts were transplantedfrom Lewis donor rats into the abdominal cavities of Fisher 344 recipients by suturing theascending aorta of donor hearts and the recipients abdominal aorta and the donor'spulmonary artery to the recipients inferior vena cava. This represents a minorhistocompatability mismatch allowing us to assess vascular changes associated with chronicrejection.4.4.1 Post-surgical morbidity and mortalityDonor and recipient rats were treated with 5 mg/kg of SP or saline control 1 h prior tosurgery in order to assess the contribution of CYP 2C to cardiac rejection. SP is a specificinhibitor of CYP 2C9 in humans and is a potent inhibitor of CYP 2C6, a putative homologue121Chapter 4of human CYP 2C9, in rats. 26 Surgical and post-surgical morbidity and mortality rates weresimilar and very low (i.e. <5%) in both SP and control groups. Post-surgical recovery wasalso similar in both groups with recipients regaining their pre-surgical weigh by 9.2 ± 2.9days in the SP group versus 10.3 ± 4.0 days in the control group (Figure 4.1). Transplantrecipients were euthanized and organs were harvested at days 4, 7 and 30 post-transplant. Atharvesting, all transplanted hearts had palpable heart beats.4.4.2 Assessment of CYP 2C6 expression in rat heart cross-sectionsInitially, immunohistochemical studies were performed to confirm expression of CYP2C6 in the transplanted hearts. Results demonstrate positive staining for CYP 2C6 in both themyocardium and vasculature of transplanted hearts (Figure 4.2).4.4.3 CYP 2C contributes to luminal narrowing in rat heterotopic heart transplantsTransplanted hearts harvested at day 30 were then utilized to assess thedevelopment of CAV by measuring the degree of luminal narrowing in the large coronaryblood vessels (Figure 4.3). Pre-treatment with SP at the time of transplantation resulted in adramatic 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 infiltrationThirty day post-transplant hearts were then assessed for characteristics of generalimmune rejection. H&E-stained sectioned were scored for diffuse, focal, sub-epicardial, sub-endocardial infiltration (Figures 4.5 and 4.6). SP pre-treatment did not result in a statistically122-11-SP (5 mg/kg)800^5^10^15^20^25^30Days post-transplant130' ± ControlChapter 4Figure 4.1 SP treatment does not reduce post-transplant weight gain.Rats receiving heterotopic heart transplants were weighed prior to transplantation and dailyfor 30-days post-transplant. Both SP and control groups had characteristic weight lossimmediately following surgery followed by gradual weight gain. Values expressed as mean ±SD (n=5). There was no difference between treatment groups (p>0.1).123Blood vessels MyocardiumC00a)0)a)zChapter 4Figure 4.2 CYP 2C6 is expressed in transplanted rat heart blood vessels andmyocardium.Paraffin-embedded transplanted hearts from Lewis rats sacrificed 30 days post-surgery wereimmunohistochemically stained for the presence of CYP 2C6 using Vector NovaRED as asubstrate. Primary antibody-absent negative staining controls and CYP 2C6 antibody-positive staining result are shown for transplanted hearts. Scale bar equals 100 vim.124Chapter 4Untreated^SP (5 mg/kg) = 100 [tmFigure 4.3 SP administration at time of surgery attenuates allograft luminal narrowing.Representative coronary blood vessels in Movat's pentachrome stained coronary crosssections from rat heterotopic heart transplants 30 days post-transplantation.125Chapter 410080-c0^60co 40 -z0 ^20-No treatment 5 mg/kg SPFigure 4.4 SP administration at time of surgery attenuates allograft luminal narrowing.Percent luminal narrowing in rat heterotopic heart transplants were performed between Lewisdonor and Fisher recipient rats 30 days post-transplantation. Luminal narrowing wasmeasured using Image-Pro Plus as the percent of the area of the lumen divided by the area ofthe internal elastic lamina. Data are expressed as mean ± SD (n= 5 hearts, 3 sections/heart, 7vessels/section, * p<0.005).126Chapter 4CHI .1 00 pmFigure 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 infiltrationand (C) focal immune myocardial infiltration.127Transplanted heartNative heart4rdz-^1p'jetkt4.^P f'11)(^k sitit 4%, 4 , P4P^147:40-,147 ••E1E500E1E)0wChapter 4H = 100 pmFigure 4.6 Histological features of epicardial and endocardial immune infiltration.Representative H&E-stained epicardial and endocardial sections from native and 30-day postsurgical heterotopic transplanted hearts. Transplanted hearts demonstrate subepicardial andsubendocardial immune infiltration.128Chapter 4significant decrease in any of these parameters (Figure 4.7). A trend was observed towards adecrease in both the number of blood vessels with perivascular infiltration and the severity ofthe infiltration but, again, these findings were not significant (p>0.1; Figure 4.8).Perivascular infiltration was not associated with luminal narrowing with some vesselsdemonstrating severe perivascular infiltration with minimal luminal narrowing and othersdemonstrating luminal narrowing with minimal infiltration (Figure 4.9).4.4.5 CYP 2C does not contribute to T-lymphocyte infiltrationT-lymphocyte infiltration was further assessed by IHC staining and quantification inboth 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 pre-treatment did not alter the degree of CD3 + or CD8+ lymphocyte infiltration in either thevascular wall (CD3+: 3.4 ± 3.6 vs. 2.0 ± 1.2 cells/100 [tm2 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.m2 for control, p=0.42; CD8+: 6.6 ± 1.4 vs. 6.4 ± 2.2 cells/100 ii.m2 for control,p=0.92).4.4.6 CYP 2C does not significantly alter post-transplant apoptosisEarly post-transplant apoptotic events were assessed by measuring TUNEL positivity in themyocardium, endothelium and smooth muscle layers of transplant sections isolated 4 dayspost-transplantation. TUNEL positivity was lower in SP treated rats in all regions assayedhowever these differences were not statistically significant (Figure 4.12). Endothelia werestained with Von Willebrand Factor and no loss of endothelial cells was detected (Figure4.13).129O No treatment®10 NM SPDiffuse^Focal^Epicardial^EndocardialFigure 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 cardiacmuscle. Data are represented as mean ± SD. There was no significant difference between SPand control groups in any immune infiltration category.130Chapter 4100 80-60-40 -20 -BChapter 4543210ANo treatment^5 mg/kg SPNo treatment^5 mg/kg SPFigure 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 cardiacmuscle. (B) The percent of blood vessels with immune infiltration is shown. Percent luminalnarrowing expressed as mean ± SD (n= 5 hearts, 7 vessels/heart) p>0.1.131Chapter 4CFigure 4.9 Perivascular immune infiltration in the absence of luminal narrowing.H&E-stained, paraffin-embedded sections from 30-day post-transplant rats showing coronaryblood vessels. (A) Representative image of native blood vessel. (B) Representative image ofCAV associated luminal narrowing in control groups. This blood vessel shows significantluminal narrowing with only moderate immune infiltration. (C) Representative image ofblood vessel showing severe perivascular immune infiltration in the absence of luminalnarrowing. Scale bar equals 100 p.m.132Vascular Wall^PVSChapter 4E 1412-OO10CD45% 80 642CO 0^ No treatment0 SP (5 mg/kg)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 ofCD3+ cells was quantified using Image-Pro Plus as the number of positive stained cells perarea of the vascular wall or the PVS. PVS was defined as the area from the outside of thedefined smooth muscle layer to the commencement of the myocardium. Data are representedas the mean ± SD of n=5 rats/group, n=7 vessels/rat.133Chapter 4^ No treatment0 SP (5 mg/kg)\iE 10- 9o 87›- , 65_CCL 4>, 32co0 10 0Vascular Wall PVSFigure 4.11 SP treatment does not alter CDS' lymphocyte infiltration in the vasculature.Paraffin-embedded sections were immuno-stained for CD8 + lymphocytes. The number ofCD8+ cells was quantified using Image-Pro Plus as the number of positive stained cells perarea of the vascular wall or the PVS. PVS was defined as the area from the outside of thedefined smooth muscle layer to the commencement of the myocardium. Data are representedas the mean ± SD of n=5 rats/group, n=7 vessels/rat.134Chapter 47— 6w= 5 -ca)> 4 -wo 3 -O.- JIII 2 -zz1- 1-:,00Myocardium^Endothelium^Smooth muscleFigure 4.12 CYP 2C does not significantly contribute to post-transplant apoptosis.Paraffin-embedded section from heterotopically transplanted hearts 4 days post-transplantwere assessed by TUNEL staining. The percentage of positively stained nuclei relative tototal nuclei relative to endothelial, smooth muscle and myocardial cells was measured usingImage-Pro Plus. No statistically significant difference between untreated and SP treatedtransplanted hearts was observed. For endothelial and smooth muscle cell counts wereperformed on 10 blood vessels/heart, 5 hearts/ group; and for myocardial measurements wereperformed on 10 fields of view/heart, and 5 hearts/group. Data are expressed as the mean ±SD.135Chapter 4H=100 pmFigure 4.13 CYP 2C-inhibition has an insignificant effect on EC loss at day 4 post-transplant.Paraffin-embedded section from heterotopically transplanted hearts 4 days post-transplantwere assessed by immuno-staining with the endothelial marker von Willebrand Factor andDAB staining. Endothelial loss was not observed in control hearts (A) or those treated withSP (13).136Chapter 44.4.7 CYP 2C contributes to SMC proliferation following transplantationSMC proliferation was assessed at day 4 following transplantation by counting cellswith 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 ofKi-67 staining (A) and representative positive staining (B).4.4.8 Peripheral cytokine and chemokine levels following transplantationSerum which was isolated at 1 day prior to transplantation and at days 1, 3, 5 and 7post-transplantation was utilized to analyze cytokine levels in the systemic circulation via theLuminex multiplex assays. Serum levels of granulocyte macrophage colony-stimulatingfactor (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 wereassessed for alterations with time and with time by group. We observed a high level ofvariability between samples. Quantified cytokine levels are shown in Table 4.1 and asummary table to statistical analyses is shown in Table 4.2. GRO/KC and IL-la, 10, 2, 10and 18 did not show any significant alterations with time or by group (p>0.1). MCP-1 levelsshowed a non-significant increase following transplantation (p=0.064) which was similar inboth 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 SPtreatment (p=0.239). INF-y levels (Figure 4.15) increased following transplantation in thecontrol group (p=0.023) however this trend was significantly reduced in the SP treated rats(p=0.028).137Chapter 4A 12C.) 10C-U)00-CO No treatment^SP (5 mg/kg)BFigure 4.14 CYP 2C contributes to SMC proliferation.Paraffin-embedded section from heterotopically transplanted hearts 4 days post-transplantwere 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 thetotal 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 Ki-67 staining (arrows). Scale bar represent 1001,1m.138Chapter 4cyto-kine(Willi) - 1Days post-transplantationCNT SP CNT SP CNT SP CNT SP CNT SPGM- 225± 317± 197± 114± 345± 162.3± 230± 459± 131± 79±CSF 211 217 122 58 233 48.1 109 297 227 93GRO/ 788 ± 808 ± 1331± 869 ± 956 ± 601± 789± 675 ± 79 7± 512 ±KC 103 59 1050 162 386 131 156 114 459 190IFN-y 21 ± 23 ± 18 ± 4 ± 52 ± 4 ± 209± 84 ± 461± 1059±9 9 21 7 56 6 258 46 813 2576MCP- 218± 183 ± 938 ± 714 ± 870 ± 1231 ± 600 ± 1294 ± 445 ± 486 ±1 101 40 350 147 559 1866 262 1961 332 377I1-la 43± 62± 52± 13± 50± 21± 72± 51± 127± 191±32 32 6 15 44 18 92 74 203 46111-113 45± 31± 23± 8± 49± 9± 26± 34± 21± 26±43 13 20 4 44 2 25 25 24 3611-2 747 ± 696 ± 448 ± 245 ± 922 ± 427 ± 653 ± 1451 ± 485 ± 312 ±843 480 230 113 676 108 455 973 627 365I1- 7± 18± 75± 7.5± 23± 423± 267± 314± 1406± 676±12p70 6 10 115 11 17 139 319 402 2425 113111-18 211± 143± 136± 98± 1486± 1017± 315± 752± 128± 207±153 28 81 51 1538 1846 170 1139 133 163Table 4.1 Peripheral cytokine and chemokine levels following heterotopic hearttransplantation 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 usingthe Luminex assay. Data are shown as mean ± SD in pg/ml for n=5 rats per group.139Chapter 4CytokineWilks' Lambda MultivariateEffect with timeTest (p-value)Effect of time by groupGM-CSF 0.004* 0.239GRO/KC 0.068 0.154IF'N-y 0.023* 0.028*MCP-1 0.064 0.775Il- 1 a 0.194 0.557I1-113 0.602 0.44911-2 0.315 0.78811-18 0.301 0.667Table 4.2 Repeated measures analysis of peripheral cytokine and chemokine levelsfollowing 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 usingthe Luminex assay. Data were analyzed for significant differences utilizing repeated measuresmultivariate analysis. P-values were calculated using the Wilks' Lambda test. (*=p< 0.05)140Chapter 4500'-6-5 400.sa.*--- 300'?.....1^■Z 200'u_1001^i its^fa h . ii .^R. ^.^!I •-2 --1100-^1^2 3 4 5 6 7 8Figure 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 Luminexassay. Data were analyzed for significant differences utilizing the repeated measuresmultivariate analysis. P-values were calculated using the Wilks' Lambda test. IFN-y levelsincrease with time (p-=0.023) and are significantly reduced in the SP treatment groupcompared to control (p=0.028).700 ■ No treatment600.^ • SP (5 mg/kg)--"Days Post-transplant141Chapter 44.4.9 CYP 2C contributes to serum 8-isoprostane levelsSerum isolated at sacrifice (day 4 or 7) was analyzed by EIA to assess oxidative stressvia free 8-isoprostane levels. Results, shown in Figure 4.16, demonstrated a peak in 8-isoprostane 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 levelswere reduced by day 7 and were similar in both groups (466 ± 62 for control vs. 543 ± 197pg/ml for SP, mean ± SEM, p=0.389).4.5 DiscussionThe findings of this study demonstrate, for the first time, that CYP 2C contributes toperi-transplant ischemic injury. We have found that inhibition of CYP 2C by SP reducesearly signs of ischemic injury including oxidative stress and a trend towards a decrease inapoptosis. This finding is consistent with our earlier studies that showed a decrease invascular and myocardial superoxide production following I/R injury following pre-treatmentwith SP.21. 25 CYPs have been previously been shown to significantly contribute to thecellular production of ROS such as 02 ., hydrogen peroxide and hydroxyl radicals during theCYP reaction cycle when electrons for the reduction of the central heme iron are transferredto the activated bound oxygen molecule. 18We also found a reduction in early (day 4) smooth muscle cell proliferation, asindicated by Ki-67 staining, in rats pre-treated with SP. This corresponds to a significant142Chapter 435003000g.. 2500a)o.2000caro2 1500o.o.0_ 1000cO5000 Day 4^ Day 7Figure 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. Serumwas analyzed for free 8-isoprostane by ETA. Data represent mean ± SD for n=5 rats/group,samples were run in triplicate.143Chapter 4decrease in intimal thickening and luminal narrowing by day 30 post-transplantation. It waspreviously hypothesized that SP increases the bioavailability of nitric oxide (NO.) byreducing its reaction with superoxide. In support of this hypothesis, SP is able to increaseNO-mediated vasodilation following ischemia/reperfusion injury. 25 Studies by Fleming'sgroup demonstrated that SP enhances endothelium-dependent vasodilator responses inpatients with manifest coronary artery disease. The observed effect was suggested to be dueto the increased bioavailability of NO- as a consequence of reduced CYP 2C9-mediatedsuperoxide generation and ONOO- formation in endothelial cells. 20 Both reduced NO andelevated ONOO- have important implications in the pathogenesis of CAV and smoothmuscle cell proliferation. Endothelial NO- is known to inhibit smooth muscle cellproliferation.27-3° We also observed an increase in IFN-y levels following transplantation inthe 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 3-kinase dependent manner. 31In summary, CYP 2C inhibition during the peri-transplant period preventeddevelopment of CAV by inhibiting early SMC proliferation and intimal hyperplasia. Thisaffect could be a result of reduced post-ischemic oxidative damage, contributing to increasedNO 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 studieswill be geared towards further elucidating the mechanism of CYP 2C-mediated post-ischemic endothelial dysfunction.144Chapter 44.6 Bibliography1. Dong C, Granville DJ, Tuffnel CE, et al. Bax and apoptosis in acute and chronicrejection of rat cardiac allografts. Lab Invest. Dec 1999 ;79(12) :1643-1653 .2. Yeung AC, Davis SF, Hauptman PJ, et al. Incidence and progression of transplantcoronary artery disease over 1 year: results of a multicenter trial with use ofintravascular ultrasound. Multicenter Intravascular Ultrasound Transplant StudyGroup. J Heart Lung Transplant. Nov-Dec 1995;14(6 Pt 2):S215-220.3. Wilhelm MJ, Kusaka M, Pratschke J, et al. Chronic rejection: increasing evidence forthe importance of allogen-independent factors. Transplant Proc. Aug1998;30(5):2402-2406.4. Laskowski I, Pratschke J, Wilhelm MJ, et al. Molecular and cellular events associatedwith ischemia/reperfusion injury. Ann Transplant. 2000;5(4):29-35.5. Valantine HA. Cardiac allograft vasculopathy: central role of endothelial injuryleading to transplant "atheroma". Transplantation. Sep 27 2003;76(6):891-899.6. Day JD, Rayburn BK, Gaudin PB, et al. Cardiac allograft vasculopathy: the centralpathogenetic role of ischemia-induced endothelial cell injury. J Heart LungTransplant. Nov-Dec 1995;14(6 Pt 2):S142-149.7. Schneeberger H, Schleibner S, Inner WD, et al. The impact of free radical-mediatedreperfusion injury on acute and chronic rejection events following cadaveric renaltransplantation. Clin Transpl. 1993:219-232.8. Tilney NL, Paz D, Ames J, et al. Ischemia-reperfusion injury. Transplant Proc. Feb-Mar 2001;33(1-2):843-844.9. Waaga AM, Gasser M, Laskowski I, et al. Mechanisms of chronic rejection. CurrOpin Immunol. Oct 2000;12(5):517-521.10. Kim J, Kil IS, Seok YM, et al. Orchiectomy attenuates post-ischemic oxidative stressand ischemia/reperfusion injury in mice. A role for manganese superoxide dismutase.J Biol Chem. Jul 21 2006;281(29):20349-20356.11. Murata S, Miniati DN, Kown MH, et al. Superoxide dismutase mimetic m40401reduces ischemia-reperfusion injury and graft coronary artery disease in rodentcardiac allografts. Transplantation. Oct 27 2004;78(8):1166-1171.12. Tanaka M, Mokhtari GK, Terry RD, et al. Overexpression of human copper/zincsuperoxide dismutase (SOD1) suppresses ischemia-reperfusion injury and subsequentdevelopment of graft coronary artery disease in murine cardiac grafts. Circulation.Sep 14 2004;110(11 Suppl 0:11200-206.13. Nelson SK, Gao B, Bose S, et al. A novel heparin-binding, human chimeric,superoxide dismutase improves myocardial preservation and protects from ischemia-reperfusi on injury. J Heart Lung Transplant. Dec 2002 ; 21(12):1296-1303.14. Yin M, Wheeler MD, Connor HD, et al. Cu/Zn-superoxide dismutase gene attenuatesischemia-reperfusion injury in the rat kidney. J Am Soc Nephrol. Dec2001;12(12):2691-2700.145Chapter 415. Abunasra HJ, Smolenski RT, Morrison K, et al. Efficacy of adenoviral gene transferwith manganese superoxide dismutase and endothelial nitric oxide synthase inreducing ischemia and reperfusion injury. Eur J Cardiothorac Surg. Jul2001;20(1):153-158.16. Wang X, Zuckerman B, Pearson C, et al. Maternal cigarette smoking, metabolic genepolymorphism, and infant birth weight. Jama. Jan 9 2002;287(2):195-202.17. Wang XL, Greco M, Sim AS, et al. Effect of CYP1A1 MspI polymorphism oncigarette smoking related coronary artery disease and diabetes. Atherosclerosis. Jun2002;162(2):391-397.18. Fleming I, Michaelis UR, Bredenkotter D, et al. Endothelium-derived hyperpolarizingfactor synthase (Cytochrome P450 2C9) is a functionally significant source ofreactive oxygen species in coronary arteries. Circ Res. Jan 19 2001;88(1):44-51.19. Fisslthaler B, Popp R, Kiss L, et al. Cytochrome P450 2C is an EDHF synthase incoronary arteries. Nature. Sep 30 1999;401(6752):493-497.20. Fichtlscherer S, Dimmeler S, Breuer S, et al. Inhibition of cytochrome P450 2C9improves endothelium-dependent, nitric oxide-mediated vasodilatation in patientswith coronary artery disease. Circulation. Jan 20 2004;109(2):178-183.21. Granville DJ, Tashakkor B, Takeuchi C, et al. Reduction of ischemia and reperfusion-induced myocardial damage by cytochrome P450 inhibitors. Proc Natl Acad Sci U SA. Feb 3 2004;101(5):1321-1326.22. Yasar U, Bennet AM, Eliasson E, et al. Allelic variants of cytochromes P450 2Cmodify the risk for acute myocardial infarction. Pharmacogenetics. Dec2003;13(12):715-720.23. Wu S, Chen W, Murphy E, et al. Molecular cloning, expression, and functionalsignificance of a cytochrome P450 highly expressed in rat heart myocytes. J BiolChem. May 9 1997;272(19):12551-12559.24. 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. JBiol Chem. Feb 16 1996;271(7):3460-3468.25. Hunter AL, Bai N, Laher I, et al. Cytochrome p450 2C inhibition reduces post-ischemic vascular dysfunction. Vascul Pharmacol. Oct 2005;43(4):213-219.26. Kobayashi K, Urashima K, Shimada N, et al. Selectivities of human cytochrome P450inhibitors toward rat P450 isoforms: study with cDNA-expressed systems of the rat.Drug Metab Dispos. Jul 2003 ;31(7):833-836.27. Tanner FC, Meier P, Greutert H, et al. Nitric oxide modulates expression of cell cycleregulatory proteins: a cytostatic strategy for inhibition of human vascular smoothmuscle cell proliferation. Circulation. Apr 25 2000;101(16):1982-1989.28. Ruiz E, Del Rio M, Somoza B, et al. L-Citrulline, the by-product of nitric oxidesynthesis, decreases vascular smooth muscle cell proliferation. J Pharmacol Exp Ther.Jul 1999;290(1):310-313.29. Janssens S, Flaherty D, Nong Z, et al. Human endothelial nitric oxide synthase genetransfer inhibits vascular smooth muscle cell proliferation and neointima formationafter balloon injury in rats. Circulation. Apr 7 1998;97(13):1274-1281.30. Stein CS, Fabry Z, Murphy S, et al. Involvement of nitric oxide in IFN-gamma-mediated reduction of microvessel smooth muscle cell proliferation. Mol Immunol.Sep 1995;32(13):965-973.146Chapter 431. Wang Y, Bai Y, Qin L, et al. Interferon-gamma induces human vascular smoothmuscle cell proliferation and intimal expansion by phosphatidylinositol 3-kinasedependent mammalian target of rapamycin raptor complex 1 activation. Circ Res. Sep14 2007;101(6):560-569.147Chapter 5: Cytochrome p450 2C9 in Vascular CellDeath and Oxidative Stress45.1 IntroductionCYPs are membrane-bound heme-containing terminal oxidases that exist in a multi-enzyme system that includes a FAD/FMN-containing NADPH cytochrome p450 reductaseand cytochrome b5. The CYP superfamily is responsible for the oxidation, peroxidationand/or reduction of vitamins, steroids, cholesterol, xenobiotics and the majority ofcardiovascular drugs in an oxygen and NADPH-dependent manner. CYP 2C9 is a mono-oxygenase that is strongly expressed in the liver and small intestine) but is also expressed inthe heart and the vasculature. CYP 2C9 is an epoxygenase and is involved in the metabolismof arachidonic acid (AA) into epoxyecosotrienoic acids (EETs). Specifically, CYP 2C9produces 5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET which are involved in NO-independent vasodilation. 2During ischemia increased intracellular calcium levels induces the activation ofphospholipase A2 (PLA2) and the subsequent hydrolysis of AA from membranephospholipids. 3-6 AA metabolism is further increased during reperfusion.7 AA is metabolizedby three major pathways; the cyclooxygenase (COX) pathway, the lipoxygenase (LOX)pathway, and the CYP epoxygenase pathway, described in detail in section 1.5. AAmetabolism during UR has been implicated as a key contributor in the progression ofischemic injury as inhibition of PLA2 or inducible PLA2 (iPLA2) protects against I/R.5' 8, 91484 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 andreoxygenation in human umbilical venous endothelial cells.Chapter 5However, several AA metabolites, including cyclooxygenase-2 (COX-2)-derivedprostacyclin, are known to be cardioprotective. CYP 2C are the primary epoxygenasesinvolved in AA metabolism by the third pathway. 1° As previously described, CYP 2C alsomake a significant contribution to the cellular production of ROS such as O2', hydrogenperoxide and hydroxyl radicals during AA metabolism. 11 ' 12Previous studies by our group and others have shown that CYP 2C9, or the rodent orrabbit equivalent, plays a role in vascular function in coronary flow following WR injury 13vasomotion 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 alsocontribute to post-transplant oxidative stress, smooth muscle proliferation and CAVdevelopment. As AA can be metabolized by one of three possible mechanisms, it is logicalthat if one of these pathways were blocked, that this may result in a shift towards the other 2pathways and increased activity of these pathways.5.2 AimWe hypothesized that CYP 2C9 expression would increase EC death and dysfunctionfollowing hypoxia and re-oxygenation (H/R). Further, that CYP 2C9 inhibition would reducethese effects whereas COX inhibition would exacerbate them. The aim of this research is toexamine the effect of CYP 2C9 in cultured human endothelial and smooth muscle cells inresponse to H/R. Specifically, we examined the contribution of CYP 2C9 to cell death in149Chapter 5endothelial and smooth muscle cell lines. We also examined the effect of COX-2 inhibitionon CYP 2C9-mediated oxidative stress following H/R.5.3 Materials and Methods5.3.1 Cell culturePooled HUVECs were obtained from Cambrex (Baltimore, MD). HUVECs werecultured in complete endothelial growth medium (EGM: endothelial basal mediumsupplemented 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 musclegrowth 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 2C9Cells were washed twice with ice cold PBS and lysed in CellLytic M lysis buffercontaining protease inhibitor cocktail (Sigma-Aldrich, Oakville, ON). Protein concentrationswere 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 CoomassieBrilliant 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 gel150Chapter 5electrophoresis and then transferred to nitrocellulose membranes. Equal volume amounts ofpurified human liver microsome (HLM) diluted 1:20 in lysis buffer was used as a control forCYP 2C9 expression. After blocking with 2% skim milk, the membranes were incubatedwith primary antibodies (1:500 anti-human CYP 2C9 (BD Gentest, San Jose, CA) or 1:1000monoclonal 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 —conjugatedsecondary antibodies (Rockland Inc. Gilbertsville, PA). Protein expression was detected byusing the Odyssey Infrared Imaging System from LI-COR Biosciences (Lincoln, NE).5.3.3 Adenoviral expression of CYP 2C9 in HUVECAn adenoviral vector expressing CYP 2C9 sense and an adenoviral vector expressingCYP 2C9 antisense, control, were kindly provided by Dr. Ingrid Fleming. Adenoviralinfections were carried out by removal of media and addition of a 10:1 virus to cell ratio inlow volume media. Cells were incubated with intermittent gentle rocking for 2 h, medialevels were restored and cells were incubated overnight to allow for protein expression.5.3.4 Optimization of hypoxic conditionsHypoxic 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 duringcardiac ischemia. 16-20 The P02 of the air and of culture media were assessed under151Chapter 5humidified conditions at 37°C and 5% CO2. Media was bubbled with chamber air for 20 sprior to measurements. P0 2 was measured using Oxylab P02 probes and a tissue oxygenationmonitor (Oxford optronix, Oxford, UK).5.3.5 Cell viability in response to H/RHUVECs transduced with Ad-CYP 2C9 sense or antisense and HCASMCs wereseeded in 96-well plates, grown to 90% confluency. Cells were pre-treated with 10 1.1Msulfaphenazole (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 washedtwice in PBS. Basal media bubbled in low oxygen conditions was then added to cells andcells were exposed to 24 h hypoxia in followed by 4 h of re-oxygenation in normoxicconditions, complete media containing 5% FBS. Viability was assessed using theCellTiter96TM AQueous Assay (MTS) (Promega, Madison, WI). MTS is described in section2.2.6. MTS was protected from light and was added at a 1:5 ratio of MTS: media and thereaction was allowed to proceed for 1 h at 37°C. Samples were measured in triplicate forabsorbance at 490 nm on the Tecan GENios Rainbow absorbance plate reader (Tecan, SanJose, CA). Data are shown as the mean ± SD and represent 3 samples per experiment for 4experiments measured in triplicate.5.3.6 Measurements of 8-isoprostaneFree 8-isoprostane measurements were performed on conditioned medium from cellculture 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 modification152Chapter 5that 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 mMaspirin were included (Cayman). Upon collection of conditioned media, 0.005% BHT(Butylated hydroxytoluene) was added to prevent further production of 8-isoprostanes in thesamples. 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, samplesand 8-isoprostane standards were incubated with EIA Buffer, AChE tracer and 8-isoprostance antiserum on the EIA plate for 18 h at room temperature. EIA plates weredeveloped by the addition of Ellman's reagent and 8-isoprostane tracer followed byincubation for 90 min at room temperature. Samples were measured via absorbance readingsat 412 nm on the Tecan GENios Rainbow absorbance plate reader (Tecan, San Jose, CA).5.4 Results5.4.1 Native, adenoviral, and H/R-induced expression of CYP 2C9 in HUVECs.Hypoxic conditions were optimized by measuring the P02 at varying oxygenconcentrations in both the chamber air and bubbled media. Results, Figure 5.1, showed thatwe were able to obtain a P02 of approximately 10% by presetting our oxygen concentrationto 1%. Measured P02 levels were comparable in the chamber air and in bubbled mediaindicating that our method of bubbling media was sufficient to control solution P02 levels. Itshould be noted that these measurements were repeated while both increasing and decreasing153Chapter 525 •20 -EC•10 10 -a.5-• P02 atmosphere0 P02 solution000^0.5^1.0^1.5^2.0^2.5^3.0% 02 Hypoxia ChamberFigure 5.1 Relationship between hypoxic chamber oxygen concentration and measuredP02.Oxygen concentrations were controlled using a hypoxic glove box. The P02 of the air and ofbubbled culture media were assessed under humidified conditions at 37°C and 5% CO2. P02was measured using Oxylab P02 probes.154Chapter 5the chamber oxygen concentration and with a second P02 probe. We did not detect anyvariability 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-CYP2C9 sense but not CYP 2C9 antisense expressed CYP 2C9, Figure 5.2(A). Hypoxia for 24 hfollowed 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 HUVECHUVECs were transduced with Ad-CYP 2C9 sense or Ad-CYP 2C9 antisense wereexposed to H24/R4 or normoxic conditions (control). Cell viability was measured using theMTS assay, shown in Figure 5.3. Cells transduced with antisense demonstrated a decrease inviability following H24/R4 to 71.3 ± 0.3% of control. These results are comparable to thosewe routinely observe in non-transduced cells. Cells transduced with sense CYP 2C9demonstrated a significantly greater drop in viability to 56.0 ± 1.9% of control (p<0.005compared to antisense cells). Pre-treatment with SP did not significantly increase viabilitypost-H24/R4 treatment in antisense transduced cells (83.0 ± 13.9% SP treated compared to71.3 ± 0.3% untreated, p>0.1). Pre-treatment with SP did significantly increase viabilitypost-H24/R4 treatment in sense transduced cells (77.0 ± 18.7% SP treated vs. 56.0 ± 1.9%untreated, p<0.05).155Chapter 5ACYP 2C9 (56 kDa)0-actin (43 kDa)BNorrnoxi4 wimior 11111111110.0 CYP 2C9(3-actinCYP 2C9H/R0-actinNon-transduced^Ad-CYP 2C9senseFigure 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 andsense. Human liver microsomes (HLM) are used as a positive control for CYP 2C9expression. (B)Non-transduced and CYP 2C9 sense transduced cells were exposed to 24 hhypoxia followed by 4 h reperfusion and normoxic time controls. CYP 2C9 expression wasanalyzed by Western blot.156Chapter 5 0 NormoxicB H/RUntreated 10 pM SP^Untreated^10 pM SPAd-CYP 2C9antisenseAd-CYP 2C9senseFigure 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 followedby 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).157Chapter 55.4.3 SP treatment does not alter SMC viability following H/R.HCASMCs were pretreated with SP and exposed to H24/R4; the results are shown inFigure 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 inviability 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 inCYP 2C9 expressing HUVECsLevels 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\/1valdecoxib, 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 toH24/R4. 8-isoprostane levels were measured using the 8-isoprostane EIA, Figure 5.5. In theabsence or presence of each inhibitor Ad-CYP 2C9 sense cells had higher levels of 8-isoprostanes in the condition media than Ad-CYP 2C9 antisense cells. For example, in theabsence 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-isoprostanelevels 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 to112.3 ± 8.4 pg/ml.158Chapter 5T.). 120Co 100V`15 80›ft.13 60'5 40CG) 20NormoxicO Untreated• 10 pM SPH/RFigure 5.4 SP treatment in HCASMCs does not alter proliferation or cell viabilityfollowing H/R.Percent viability of HCASMCs exposed to 24 h hypoxia followed by 4 h reperfusion andnormoxic time controls. Cells were either untreated or treated with 10 µM SP 1 h prior to theinduction of hypoxia. Values are expressed as mean ± SE (n=6, * p<0.05).159o Anti-sensen Sense140 -12010080604020Chapter 5No Inhibitor^Sulfaphenazole^Valdecoxib^AspirinFigure 5.5 CYP 2C increases 8-isoprostane levels.HUVECs transduced with Ad-CYP 2C9 sense or antisense and HCASMCs were pre-treatedwith SP, valdecoxib or aspirin 1 h prior to induction of H/R. Cells were exposed to 24 hhypoxia in followed by 4 h of re-oxygenation in normoxic conditions. Free 8-isoprostanemeasurements were performed on conditioned medium by EIA. Data represents mean ± SD,n=3, * p<0.05).160Chapter 5Aspirin treatment showed little effect on 8-isoprostane levels in CYP 2C9 expressingHUVECs.5.5 DiscussionOur previous studies have shown an important role for CYP 2C in post-ischemicvascular function and CAV development.(15, Chapter 4) In both of these studies it is hypothesizedthat 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 CYP2C9 in cultured endothelial and smooth muscle cells following exposure to H/R, as an invitro model of I/R.CYP 2C9 is expressed in human endothelial cells. 21 However, upon culturing, CYP2C9 levels in endothelial cells rapidly decrease both at the mRNA and protein level. 22' 23Therefore, our finding that CYP 2C9 is not expressed in cultured HUVECs was notunexpected. In order to examine the role to CYP 2C9 in these cells we employed adenoviralvectors expressing sense and anti-sense (control) CYP 2C9. Previous studies have shown thatCYP 2C mRNA and protein levels increase following hypoxia. 22 We examined whetherexposing 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 than2C9 are responsible for this induction.We have previously shown that the CYP 2C9 inhibitor SP protects againstendothelium-dependent vascular dysfunction following I/R in rats. 15 Therefore, we wanted toexamine whether CYP 2C9 would also contribute to endothelial cell death and dysfunction in161Chapter 5cultured endothelial cells. Our results indicate that CYP 2C9 expression results in increasedcell death following exposure to H/R. Pre-treatment with SP is able to protect against H/R inHUVECs expressing CYP 2C9 sense but not anti-sense. We also found that CYP 2C9 senseexpressing cells produce increased oxidant radicals, as indicated by free 8-isoprostane levelsin the conditioned media, following H/R and that SP was able to reduce 8-isoprostane levelsin these cells.We have previously found that SP treatment during I/R reduces subsequent SMCproliferation following heterotopic heart transplantation in rats, see Figure 4.13. Although itis likely that this resulted from decreased bioavailability of endothelium derived NO and notfrom the single dose of SP given prior to transplantation, we examined the possibility that SPwas directly affecting SMCs. We detected only a slight decrease in the number of viableSMCs following SP treatment. H/R cause a decrease in cell viability in both untreated and SPtreated groups and exposure to H/R did not increase or decrease this effect. This result agreeswith our previous finding that SMC-dependent relaxation is impaired following I/R and thatSP pre-treatment is not protective against this type of vascular dysfunction. 15As described in section 1.5.4, studies related to celecoxib and paracoxib/valdecoxibhave also shown an association with increased cardiovascular risks resulting in thewithdrawal of paracoxib/valdecoxib from the market. 24-26 Given that increased AA liberationis associated with ischemia, it is possible that under conditions of ischemia, in the presenceof coxibs, that AA liberation would stimulate elevated CYP 2C activity and subsequent ROSproduction. This elevation in ROS production may result in vascular injury and dysfunctionthat could contribute to the problems associated with coxib administration in the presence ofischemia. Therefore, we were interested in whether COX inhibition could induce increased162Chapter 5oxidative damage by CYP 2C9. We hypothesized that decreased AA metabolism by COX-2would lead to increased AA metabolism by CYP 2C9 and consequently increased oxidativestress. Our results show that valdecoxib treatment did increase 8-isoprostane formation inCYP 2C9 sense but not anti-sense expressing cells. However, aspirin, administered at a dosespecific for COX-1 did not alter 8-isoprotane production in either CYP 2C9 expressing orcontrol cells. Thus, it is not clear whether altered AA metabolism is involved. One factorcomplicating this analysis is that valdecoxib, like most COX-2 inhibitors, is largelymetabolized via CYP 2C9. 27 Studies have not been performed to assess whether low dosevaldecoxib can induce CYP 2C9 activity although high dose valdecoxib, IC50 41 ILLM, acts asa moderate inhibitor of 2C9 (Bextra label information, Pfizer Inc. NY, NY).In conclusion, we demonstrate that CYP 2C9 expression in endothelial cells leads toincreased cell death and ROS production following H/R and that pre-treatment with the CYP2C9 inhibitor SP can reduce these effects. SP did not have an effect of H/R-induced celldeath in SMC. We also present intriguing data indicating that the COX-2 inhibitorvaldecoxib induces increased oxidant stress in CYP 2C9 expressing endothelial cells. Furtherdetailed experimentation will be required to elucidate the mechanism of COX-2 inducedoxidant production and to determine if altered AA metabolism plays a role.163Chapter 55.6 Bibliography1. Bieche I, Narjoz C, Asselah T, et al. Reverse transcriptase-PCR quantification ofmRNA levels from cytochrome (CYP)1, CYP2 and CYP3 families in 22 differenthuman tissues. Pharmacogenet Genomics. Sep 2007;17(9):731-742.2. Potente M, Michaelis UR, Fisslthaler B, et al. Cytochrome P450 2C9-inducedendothelial cell proliferation involves induction of mitogen-activated protein (MAP)kinase phosphatase-1, inhibition of the c-Jun N-terminal kinase, and up-regulation ofcyclin Dl. J Biol Chem. May 3 2002;277(18):15671-15676.3. Freyss-Beguin M, Millanvoye-van Brussel E, Duval D. Effect of oxygen deprivationon metabolism of arachidonic acid by cultures of rat heart cells. Am J Physiol. Aug1989;257(2 Pt 2):H444-451.4. Leong LL, Sturm MJ, Ismail Y, et al. Plasma phospholipase A2 activity in clinicalacute myocardial infarction. Clin Exp Pharmacol Physiol. Feb 1992 ;19(2): 113-118.5. Van der Vusse GJ, Reneman RS, van Bilsen M. Accumulation of arachidonic acid inischemic/reperfused cardiac tissue: possible causes and consequences. ProstaglandinsLeukot Essent Fatty Acids. Jul 1997;57(1):85-93.6. Williams SD, Gottlieb RA Inhibition of mitochondrial calcium-independentphospholipase A2 (iPLA2) attenuates mitochondrial phospholipid loss and iscardioprotective. Biochem J. Feb 15 2002;362(Pt 1):23-32.7. Hendrickson SC, St Louis JD, Lowe JE, et al. Free fatty acid metabolism duringmyocardial ischemia and reperfusion. Mol Cell Biochem. Jan 1997;166(1-2):85-94.8. Ogata K, Jin MB, Taniguchi M, et al. Attenuation of ischemia and reperfusion injuryof canine livers by inhibition of type II phospholipase A2 with LY329722.Transplantation. Apr 27 2001;71(8):1040-1046.9. Sargent CA, Vesterqvist 0, McCullough JR, et al. Effect of the phospholipase A2inhibitors quinacrine and 7,7-dimethyleicosadienoic acid in isolated globally ischemicrat hearts. J Pharmacol Exp Ther. Sep 1992;262(3):1161-1167.10. Luo G, Zeldin DC, Blaisdell JA, et al. Cloning and expression of murine CYP2Cs andtheir ability to metabolize arachidonic acid. Arch Biochem Biophys. Sep 11998;357(1):45-57.11. Fleming I, Michaelis UR, Bredenkotter D, et al. Endothelium-derived hyperpolarizingfactor synthase (Cytochrome P450 2C9) is a functionally significant source ofreactive oxygen species in coronary arteries. Circ Res. Jan 19 2001;88(1):44-51.12. Fulton D, McGiff JC, Wolin MS, et al. Evidence against a cytochrome P450-derivedreactive oxygen species as the mediator of the nitric oxide-independent vasodilatoreffect of bradykinin in the perfused heart of the rat. J Pharmacol Exp Ther. Feb1997;280(2):702-709.13. Granville DJ, Tashakkor B, Takeuchi C, et al. Reduction of ischemia and reperfusion-induced myocardial damage by cytochrome P450 inhibitors. Proc Natl Acad Sci U SA. Feb 3 2004;101(5):1321-1326.14. Fichtlscherer S, Dimmeler S, Breuer S, et al. Inhibition of cytochrome P450 2C9improves endothelium-dependent, nitric oxide-mediated vasodilatation in patientswith coronary artery disease. Circulation. Jan 20 2004;109(2):178-183.164Chapter 515. Hunter AL, Bai N, Laher I, et al. Cytochrome p450 2C inhibition reduces post-ischemic vascular dysfunction. Vascul Pharmacol. Oct 2005;43(4):213-219.16. Carrier M, Trudelle S, Thai P, et al. Ischemic threshold during cold bloodcardioplegic 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 humanheart transplantation. A potential role for endothelial cells. Circulation. Nov 11995;92(9 Suppe:11428-432.18. Pinsky DJ, Naka Y, Liao H, et al. Hypoxia-induced exocytosis of endothelial cellWeibel-Palade bodies. A mechanism for rapid neutrophil recruitment after cardiacpreservation. J Clin Invest. Jan 15 1996;97(2):493-500.19. Wiener L, Santamore WP, Venkataswamy A, et al. Postoperative monitoring ofmyocardial oxygen tension: experience in 51 coronary artery bypass patients. ClinCardiol. Aug 1982;5(8):431-435.20. Zuurbier CJ, van Iterson M, Ince C. Functional heterogeneity of oxygen supply-consumption 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 rolein the regulation of exercise-induced skeletal muscle blood flow and oxygen uptake inhumans. J Physiol. Jan 1 2003;546(Pt 1):307-314.22. Michaelis UR, Fisslthaler B, Barbosa-Sicard E, et al. Cytochrome P450 epoxygenases2C8 and 2C9 are implicated in hypoxia-induced endothelial cell migration andangiogenesis. J Cell Sci. Dec 1 2005;118(Pt 23):5489-5498.23. Michaelis UR, Fisslthaler B, Medhora M, et al. Cytochrome P450 2C9-derivedepoxyeicosatrienoic acids induce angiogenesis via cross-talk with the epidermalgrowth factor receptor (EGFR). Faseb J. Apr 2003;17(6):770-772.24. Nussmeier NA, Whelton AA, Brown MT, et al. Complications of the COX-2inhibitors parecoxib and valdecoxib after cardiac surgery. N Engl J Med. Mar 172005;352(11):1081-1091.25. Ott E, Nussmeier NA, Duke PC, et al. Efficacy and safety of the cyclooxygenase 2inhibitors parecoxib and valdecoxib in patients undergoing coronary artery bypasssurgery. J Thorac Cardiovasc Surg. Jun 2003;125(6):1481-1492.26. Solomon SD, McMurray JJ, Pfeffer MA, et al. Cardiovascular risk associated withcelecoxib in a clinical trial for colorectal adenoma prevention. N Engl J Med. Mar 172005;352(11):1071-1080.27.^Ibrahim A, Park S, Feldman J, et al. Effects of parecoxib, a parenteral COX-2-specific inhibitor, on the pharmacokinetics and pharmacodynamics of propofol.Anesthesiology. Jan 2002;96(1):88-95.165Chapter 6: Summary and Conclusions6.1 Restatement of the ProblemMore than 3,000 heart transplants are performed annually worldwide. Currentimmunosuppressive regimens are very effective in preventing acute rejection. However,chronic rejection associated with CAV remains the major hurdle to long-term graft survivalof all vascularized organ transplants. CAV is an accelerated and diffuse form ofarteriosclerosis' that can be detected in up to 75% of heart transplant recipients following thefirst year of transplantation. 2 Although immunological mechanisms clearly play an importantrole in the pathogenesis of CAV, non-immunological mechanisms, such as peri-transplantUR injury, also contribute via direct damage or indirectly through cross-talk with immuneresponses associated with this type of vasculopathy. 3' 4There are relatively few treatments for established CAV and re-transplantation isoften the only option. Given the short supply of donor organs and the added stress of anadditional major surgery, even re-transplantation is not a viable option for many patients.Treatments to control risk factors, such as CMV infections and hyperlipidemia6, have shownthe most promising results at preventing CAV development. Given the association betweenvascular I/R injury and CAV development our primary aim was to examine whetherinhibition of peri-transplant FR injury could reduce or prevent CAV treatment. We selectedtwo targets that are known to protect against and contribute to oxidant injury, ARC and the166Chapter 6CYP 2C enzymes, respectively, to examine this link based on results shown in myocardialinfarction?' 86.2 Summary of FindingsInitially, we examined the ability to ARC to protect against oxidant stress in vascularcell types (Chapter 2). We demonstrated, for the first time, that ARC is expressed in bothcultured endothelial and smooth muscle cells by RT-PCR and immunoblotting. We thenincreased ARC levels utilizing TAT-fusion protein transduction and examined ARC's abilityto protect against oxidant-mediated cell death induced by treatment with H202. TAT-ARCdid not confer increased protection against H202 treatment than did treatment with ourcontrol protein TAT-0-gal. These results differed from results obtained in H9c2 ratembryonic cardiomyocytes. 8During our control experiments analyzing the action of ARC in H9c2 cells, weobserved that ARC overexpressing cells did not undergo differentiation induced by serumwithdrawal. We examined this observation further and demonstrated that ARC expressionlevels increase and stabilize upon differentiation in non-transduced H9c2 cells. ARC-overexpression in pre-differentiated H9c2-cells suppressed differentiation; indicated byincreased myotube formation, nuclear fusion and expression of the differentiation markersmyogenin and troponin-T. ARC-overexpression inhibited myoblast differentiation associatedcaspase-3 activation, suggesting ARC inhibits myogenic differentiation through caspaseinhibition. Thus, we have demonstrated a novel role for ARC in the regulation of muscledifferentiation.167Chapter 6As we were unable to obtain sufficient preliminary data indicating a role for ARC inprotection against oxidative damage in the vasculature, we turned our attention to the CYP2C family of enzymes. Specifically, we examined the role of the CYP 2C9-like enzyme inrodents. Our initial examinations involved assessment of vascular function followingischemia and reperfusion (Chapter 3). Previous studies have shown that vascular function isimpaired following ischemic injury 9-14 and that CYP inhibitors provide protection againstmyocardial infarction7 and vascular dysfunction in patients with manifest coronary arterydisease. 15 Therefore, we hypothesized that SP, an inhibitor of CYP 2C9, would also attenuatepost-ischemic endothelial dysfunction. We utilized the Langendorff model of I/R in rats andanalyzed vascular function in septal coronary resistance arteries by pressure myography. 16I/R caused impairment in both endothelium-dependent and independent vasodilation. Pre-treatment with SP restored endothelial sensitivity to ACh but did not restore sensitivity toendothelium-independent vasodilators. I/R-induced superoxide production was assessed bydihydroethidium staining of flash frozen hearts. SP treatment significantly reducedsuperoxide production in arterial walls following I/R injury. Therefore, we concluded thatCYP 2C contributes to impaired post-ischemic endothelium-dependent, NO-mediatedvasodilation by increasing superoxide production.Given the protective role of the CYP 2C inhibitor SP in protection against vasculardysfunction following I/R and the link between peri-transplant I/R injury, post-transplantvascular dysfunction and CAV, we explored whether CYP 2C may also contribute to theonset 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 or168Chapter 6weight gain. Assessment of coronary blood vessels from rats 30 days post-transplantindicated that treatment with SP significantly reduced luminal narrowing. However, SP didnot reduce diffuse, focal, epicardial, endocardial or perivascular immune infiltration nor didit alter infiltration by lymphocytes as measured by CD3 + and CD8+ staining. SP also did notsignificantly alter TUNEL positivity in myocardial, endothelial or SMC populations. We didnot observe endothelial loss in either the SP-treated or control groups. Analysis of rats 4 dayspost-transplant demonstrated a decrease in SMC proliferation in the SP-treated rats comparedto controls. In addition, the SP-treatment group had decreased levels of serum IFN-y and 8-soprostane post-transplantation.Our final set of experiments examined the effects of CYP 2C9 in cultured vascularcells in response to H/R. We demonstrated that HUVECs do not express CYP 2C9 followingculture and that H/R does not induce expression. We successfully induced expression of CYP2C9 through the use of adenoviral constructs and demonstrated that CYP 2C9 contributes tocell 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 ofROS. Our results indicated that the COX-2 inhibitor, valdecoxib, but not aspirin given at adose specific for COX-1, caused increased ROS production in CYP 2C9 expressing cells. Itis unclear if valdecoxib is exerting this effect through direct induction of CYP 2C9, alteredAA metabolism or an alternative pathway.169Chapter 66.3 Relevance of FindingsThe relationship between peri-transplant ischemic injury and CAV has beenpreviously described. 3' 4. 17-21 However, effective strategies for CAV by preventing ROSproduction during peri-transplant I/R have not been reported.Some previous studies have linked peri-operative ROS scavenging treatments usingantioxidants which reduced CAV development. Murata et al. 22 demonstrated that peri-operative treatment with the SOD mimetic m40401 reduced CAV development. Morerecently, Iwanaga et a1. 23 demonstrated that peri-operative treatment with the antioxidantriboflavin reduces both acute rejection and CAV development in mice. Complementarystudies have been described in renal transplantation where the use of peri-operativeantioxidants have reduced both acute and chronic obliterative arteriosclerosis. 24' 25Studies targeting the sources of ROS have been examined in the field of myocardialI/R related to infarction. As described in section 1.2.2, several candidate pathways have beenproposed 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 notmet great success. NADPH oxidase inhibition, by use of p47-null mice, revealed nosignificant difference in infarct size. 26 Xanthine oxidase inhibitors have also failed to protectagainst I/R and may be contraindicated in patients with ischemic disease 26' 27 while eNOSmay play a protective role. 28 Thus, the discovery that CYP 2C9 inhibition significantlyreduced myocardial infarction was exciting. 7Our 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 of170Chapter 6administering SP, to inhibit CYP 2C9, only during the peri-operative period demonstrated aclear link between peri-operative I/R and the development of vascular dysfunction and CAV.6.4 Future DirectionsThroughout the course of this thesis we have made several interesting and novelfindings. Each of these findings warrants further examination.With respect to our findings related to ARC in the vasculature, we were not able toshow protection against oxidative injury induce by f202. However, as ARC is expressed inthe vasculature, it is logical to assume that it is serving a function in these vascular beds andARC has been found to be a multifactorial anti-apoptotic protein. 29 Thus there are manypotential targets for ARC in these cells. Further experimentation examining alternativeinducers of apoptosis and necrosis, both oxidative and non-oxidative may uncover a role forARC in these cells. Our findings related to ARC's inhibition of myocyte differentiationindicate that the mechanism likely involves inhibition of caspase-3 activity. However, furtherexperimentation 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, alsocreate many interesting questions. If our hypothesis is correct and SP treatment protectsagainst vascular injury by increasing post-ischemic NO bioavailability, then it is not clearwhy direct addition of NO , by the NO donor SNP, did not have a similar effect. This effectwas also described by Fichtlsherer et al.. 15 It is possible that this difference reflects themechanism of NO- transfer between endothelial and SMCs. It is also possible that SP's171Chapter 6restoration of endothelium-dependent vasodilation involves other factors outside ofmaintenance of NO bioavailability, likely related to decreased oxidative damage to theendothelium. Direct examination of the role of AA release and examination of alternativeCYP 2C substrates would undoubtedly provide mechanistic insight into CYP 2C role in I/Rinjury. Experiments related to detailed examination of alterations in AA metabolism havecommenced in our laboratory. These experiments would not only provide insight into CYP2C deleterious effects during I/R but may also provide insight into why COX-2 inhibitorshave been associated with increased risk of cardiovascular events. Our finding that CYP 2Cinhibition during the peri-transplant period could have clinical importance. In order totranslate this research into a clinical setting there are several questions that should beaddressed. One intriguing possibility is that addition of CYP 2C inhibitors to cardioplegicsolutions could be sufficient to confer protection. This situation would be advantageous as itwould not require pre-treatment of donors and would likely reduce unwanted drug-druginteractions with cardiovascular drugs necessary for recipient treatment. If donor andrecipient 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 2Cfamily would also have to be considered.6.5 Concluding RemarksOur overarching goal of these studies was to examine methods of preventing or reducingvascular I/R injury and subsequent development of CAV. Although we did not obtain172Chapter 6positive results in our preliminary data related to ARC in vascular oxidative damage we diduncover a serendipitous role for ARC in myogenic differentiation. In our studies related toCYP 2C9, we were able to reduced post-ischemic oxidative stress, reduce endothelium-dependent vascular dysfunction and significantly reduce CAV development.173Chapter 66.6 Bibliography1. Dong C, Granville DJ, Tuffnel CE, et al. Bax and apoptosis in acute and chronicrejection of rat cardiac allografts. Lab Invest. Dec 1999; 79(12): 1643-1653.2. Yeung AC, Davis SF, Hauptman PJ, et al. Incidence and progression of transplantcoronary artery disease over 1 year: results of a multicenter trial with use ofintravascular ultrasound. Multicenter Intravascular Ultrasound Transplant StudyGroup. J Heart Lung Transplant. Nov-Dec 1995;14(6 Pt 2):S215-220.3. Wilhelm MJ, Kusaka M, Pratschke J, et al. Chronic rejection: increasing evidence forthe importance of allogen-independent factors. Transplant Proc. Aug1998;30(5):2402-2406.4. Laskowski I, Pratschke J, Wilhelm MJ, et al. Molecular and cellular events associatedwith i schemia/reperfu si on injury. Ann Transplant. 2000 ;5 (4): 29-35 .5. Valantine HA, Luikart H, Doyle R, et al. Impact of cytomegalovirus hyperimmuneglobulin on outcome after cardiothoracic transplantation: a comparative study ofcombined prophylaxis with CMV hyperimmune globulin plus ganciclovir versusganciclovir alone. Transplantation. Nov 27 2001;72(10):1647-1652.6. Wenke K, Meiser B, Thiery J, et al. Impact of simvastatin therapy after hearttransplantation an 11-year prospective evaluation. Herz. Aug 2005;30(5):431-432.7. Granville DJ, Tashakkor B, Takeuchi C, et al. Reduction of ischemia and reperfusion-induced myocardial damage by cytochrome P450 inhibitors. Proc Nall Acad Sci U SA. Feb 3 2004;101(5):1321-1326.8. Gustafsson AB, Sayen MR, Williams SD, et al. TAT protein transduction intoisolated perfused hearts: TAT-apoptosis repressor with caspase recruitment domain iscardioprotective. Circulation. Aug 6 2002;106(6):735-739.9. Benvenuti C, Aptecar E, Mazzucotelli JP, et al. Coronary artery response to cold-pressor test is impaired early after operation in heart transplant recipients. J Am CollCardiol. Aug 1995;26(2):446-451.10. Legare JF, Issekutz T, Lee TD, et al. CD8+ T lymphocytes mediate destruction of thevascular media in a model of chronic rejection. Am J Pathol. Sep 2000;157(3):859-865.11. Moien-Afshari F, McManus BM, Laher I. Immunosuppression and transplantvascular disease: benefits and adverse effects. Pharmacol Ther. Nov2003;100(2):141-156.12. Mugge A, Heublein B, Kuhn M, et al. Impaired coronary dilator responses tosubstance P and impaired flow-dependent dilator responses in heart transplantpatients with graft vasculopathy. J Am Coll Cardiol. Jan 1993;21(1):163-170.13. Vassalli G, Gallino A, Kiowski W, et al. Reduced coronary flow reserve duringexercise in cardiac transplant recipients. Circulation. Feb 4 1997;95(3):607-613.14. Weis M, Wildhirt SM, Schulze C, et al. Coronary vasomotor dysfunction in thecardiac allograft: impact of different immunosuppressive regimens. J CardiovascPharmacol. Dec 2000;36(6):776-784.174Chapter 615. Fichtlscherer S, Dimmeler S, Breuer S, et al. Inhibition of cytochrome P450 2C9improves endothelium-dependent, nitric oxide-mediated vasodilatation in patientswith 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 post-ischemic vascular dysfunction. Vascul Pharmacol. Oct 2005 ;43(4): 213-219.17. Land W, Messmer K. The impact of ischemia/reperfusion injury on specific and non-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 centralpathogenetic role of ischemia-induced endothelial cell injury. J Heart LungTransplant. Nov-Dec 1995;14(6 Pt 2):S142-149.19. Valantine HA. Cardiac allograft vasculopathy: central role of endothelial injuryleading 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. Feb-Mar 2001;33(1-2):843-844.21. Waaga AM, Gasser M, Laskowski I, et al. Mechanisms of chronic rejection. CurrOpin Immunol. Oct 2000;12(5):517-521.22. Murata S, Miniati DN, Kown MH, et al. Superoxide dismutase mimetic m40401reduces ischemia-reperfusion injury and graft coronary artery disease in rodentcardiac allografts. Transplantation. Oct 27 2004;78(8):1166-1171.23. Iwanaga K, Hasegawa T, Hultquist DE, et al. Riboflavin-mediated reduction ofoxidant 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 humanrecombinant superoxide dismutase on acute and chronic rejection events in recipientsof cadaveric renal transplants. Transplantation. Jan 1994;57(2):211-217.25. Vela C, Cristol JP, Maggi MF, et al. Oxidative stress in renal transplant recipientswith 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 inNADPH oxidase-deficient mice. Circ Res. Oct 27 2000;87(9):812-817.27. Parmley LF, Mufti AG, Downey JM. Allopurinol therapy of ischemic heart diseasewith infarct extension. Can J Cardiol. Apr 1992;8(3):280-286.28. Sharp BR, Jones SP, Rimmer DM, et al. Differential response to myocardialreperfusion injury in eNOS-deficient mice. Am J Physiol Heart Circ Physiol. Jun2002;282(6):H2422-2426.29.^Ekhterae D, Lin Z, Lundberg MS, et al. ARC inhibits cytochrome c release frommitochondria and protects against hypoxia-induced apoptosis in heart-derived H9c2cells. Circ Res. Dec 9 1999;85(12):e70-77.175Appendix IAppendix I: Animal Care Certificate forTransplantationhttps://rise.ubc.ca/rise/Doc/0/H7H8J5M9E6LK3BO1 . I t34AOQFLAAL.1*^THE UNIVERSITY OF BRITISH COLUMBIAANIMAL CARE CERTIFICATEApplication Number: A05-0019Investigator or Course Director: David J. GranvilleDepartment: Pathology & Laboratory MedicineAnimals:Rats F344 112Rats Lewis 205Start Date: January 2, 2005 ApprovalDate: March 22, 2006Funding Sources:Canadian Institutes of Health ResearchThe role of CYP2C9 in peri-transplant ischemic injury and transplant vasculardiseaseDean of MedicineStart Up FundingHeart and Stroke Foundation of B.C. & YukonThe role of CYP2C9 in peri-transplant ischemic injury and transplant vasculardiseaseHeart & Stroke Foundation of CanadaRole of CYP2C9 in Transplant Vascular DiseaseCanadian Institutes of Health ResearchRole of CYP2C9 in Cardiac Ischemia and Reperfusion InjurySt. Paul's HospitalCytochrome p450 monooxygenases: Role in endothelial and smooth muscle celldeath and cardiac transplant vascular diseaseGrant Agency:Grant Title:Grant Agency:Grant Title:Grant Agency:Grant Title:Grant Agency:Grant Title:Grant Agency:Grant Title:Grant Agency:Grant Title:1 of 2^ 11%29/2007 6:33 PM176Appendix Ihttps://rise.ubc.ca/rise/Doc/0/1-17H8J5M9E6LK3BOFTB4AOQFLAA , ...Grant Agency: Michael Smith Foundation for Health ResearchGrant Title:^Transplantation training programUnfunded title: CYP2C9 contributes to cardiac ischemia and reperfusion injuryThe Animal Care Committee has examined and approved the use of animals for theabove 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 theCCAC and some granting agencies.A copy of this certificate must be displayed in your animal facility.Office of Research Services and Administration102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3Phone: 604-827-5111 Fax: 604-822-50932 of 2^ 11/29/2007 6:33 PM177Appendix IIAppendix II: Rat Heterotopic Heart TransplantationSOPSOP: The technique for heterotopic cardiac transplantation in ratsSubmitted by: Alexandra KerjnerDate: 05/05/2006The technique for heterotopic cardiac transplantation in ratsPurpose: 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 staffmember proficient in the technique or have equivalent previous experience.Responsibility: Veterinarian, Technical Personnel, InvestigatorMaterials: Micro Instruments1 curved microneedle driver1 straight microneedle driver1 straight microscissor 1 45° angle microscissor 3-4 Jeweller's forceps 1 angled Jeweller's forcepsMacro Instruments 4 haemostats (1 large, 2 medium, 1 small) 1 small curved Steven scissor 1 suture scissor 1 large scissor 2 large forceps178Appendix II 1 large needle driver 1 Allis forceps 1 large curved paediatric clampThe procedure is carried out using an operating microscope at a magnification between 4 to25X.Procedure: In this model the donor ascending aorta is sutured end-to-side to the recipient abdominalaorta and the donor pulmonary artery is anastomosed to the recipient inferior vena cava(IVC). Hearts transplanted heterotopically behave functionally as aorto-caval fistulae. Bloodenters the donor, ascending aorta from the recipient abdominal aorta and is diverted into thecoronary arteries by the closed aortic valve. After the myocardium is perfused, venous blooddrains into the right atrium through the coronary sinus and is pumped back into the recipientNC by the right ventricle.Recipient^Recipient(VC^abdominal aorta/Donor ascending aortaDonor pulmonary arteryDonor operation:Drawing of the anastomoses in the heterotopiccardiac grafts. The donor ascending aorta is suturedend-to-side to the recipient abdominal aorta and thedonor pulmonary artery is anastomosed to therecipient inferior vena cava (IVC).The rat is anaesthetized with Xylazine (10 mg/kg)/Ketamine (120 mg/kg), IP. The rat isshaved, prepped with alcohol and betadine and moved to the surgery table. Sterile drapes areplaced; aseptic technique being used throughout. The donor's abdominal cavity is openedwith a large longitudinal incision long the abdomen, at the bottom of the ribs. The diaphragmand the lateral aspects of the rib cage are cut and the abdominal contexts are moved to the leftside, exposing the IVC. The IVC is isolated above the liver and a loose tie is placedproximally, the NC is clamped distally. Using a 3m1 syringe and a 25 gauge needle, the NCis cannulated and slowly perfused with heparinized saline, making sure that no air gets into179Appendix IIthe vessel. The right and left superior vena cava (SVC) are isolated and two ligatures areplaced leaving space between then. The SVC is cut between the two ligatures. The ascendingaorta is cut below the brachiocephalic artery and the main pulmonary artery is cut proximalto its bifurcation, they are flushed with heparinized saline. The connective tissue between theascending aorta and pulmonary artery is cut away at this stage. The pulmonary veins areligated together with a single 6-0 silk tie. The donor heart is gently detached and flushed withheparinized saline. At this point it might be necessary to tie off a small vessel very close tothe aorta and pulmonary artery. The heart is then weighed and placed into a heparinizedsaline bowl on ice.Superior vena caN a (3)Inferior vena ea,. (. I .)Donor preparation. Superior and inferior vena cava, azygos vein and pulmonary vein wereligated. 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 reducethe ischemic times. The animal is weighed, anaesthetized with isoflurane inhalationanaesthesia in an induction chamber set at 4% anaesthesia and prepped in the same manneras the donor. Ophthalmic ointment (Lacri-lube) is put in the eyes to prevent drying and theanimal is ear marked. A well insulated heating pad is positioned under the rat so that the180Appendix IIbody 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 retractedon 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 foranastomoses 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. Afterensuring that the orientation of the donor PA is correct, end-to-side anastomosis betweendonor PA and recipient IVC is performed using continuous 9-0 nylon sutures. The posteriorwall is sutured within the vessel lumen without repositioning the graft. The anterior wall isthen closed externally using the same suture. Once the venous anastomosis is completed, thevein 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 recipientaorta is performed in the fashion as the venous anastomosis. A small quantity ofmicrofibrillar collagen (Avitene) is placed around the arterial anastomosis before releasingthe clamp. Gentle pressure is applied to the anastomotic site with a dry cotton swab for 1-2minutes after the clamp is removed. Sites of possible leaks are checked. The bowel isreturned 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 isplaced in the paediatric incubator for a few hours with supplemental oxygen. Buprenophrineis administered subcutaneously at 0.01 mg/kg immediately following surgery. If necessaryadditional buprenophrine will be administered twice every 8-12 hours given signs ofpostoperative pain. Signs of postoperative pain include: decreased activity or a reluctance tomove, 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 inrats (Chromodacryorrhoea), unusual aggressiveness when handled.181Appendix HScoring System for Heart Transplant AnimalsThis system is designed to give an overview of the health status of experimental mice andrats involved in heart transplant projects. These animals undergo the very strenuous andstressful surgical procedure of having a heart transplanted into their abdomen. Each animal ismonitored very carefully and scored daily using this system. The animal unit's staff is alwaysavailable to give assistance and advice. Please do not hesitate to contact us if you have anyquestions or concerns.Animals are evaluated on a number of criteria and given a score from 0 to 4.Score from: 0=normal1= minimal/mild but noticeable4= moderate to severeIf the score is 4 for any criteria, consult with the animal unit group and/or designatedexperiment 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 groupand/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 willrequire intervention.NOTE: If treatment or intervention of any kind will render the experiment unusable pleaseinform animal unit staff at the onset of the experiment. If this is the case, animals whichdisplay illness or other problems will be euthanized.Attitude: 0= BAR (bright, alert/active and responsive)1= Burrowing or hiding, quiet but rouses when touched182Appendix II4= No cage exploration when lid is off, burrows/hides, head presses, may or may notvocalize or be unusually aggressive when touched, no nesting, may seem confused/irritatedor hyper responsive.Appearance: 0= normal1= mild piloerection, mild to moderate dehydration, soft stool4= severe piloerection, moderate to sever dehydration (obvious at first glance),sunken/wasted appearance, diarrhoea (moderate to severe can be smelled easily and seen onlight coat colors easily), laboured breathing, yellowing or whitish looking mucus membranes(skin) colour, animal is hot or cold to the tough.Gait/Posture: 0= normal1= mild in coordination when stimulated, slight hunched posture, slight limp4= obvious ataxia or head tilt, severe hunching, tippy-toeing, favouring of limb/noticeablelimp or paralysis of limb(s)Weight: (post surgical or post experiment)0= none or up to 10% weight loss1= 11-15% weight loss4= 16-20% weight lossAppetite: 0= normal, eats dry food, evidence of urine and feces on cage bottom, food missing fromhopper or floor, Jell-O or supplements gone after 8 hours1= no evidence of eating dry food but likes Jell-O or supplements4= no interest in food or supplements183Appendix IIIn addition to the categories which are given a score, here is a list of things which must alsobe evaluated on a regular basis. If any of these are noted, please consult with animal unit staffor 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 withoutsores 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 onexperiment and reactions. Have others check your experimental animals occasionallyto minimize bias.*** If any treatments are indicated they must be approved beforehand by the PI in order toassure 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 madeeach time the animal is evaluated. Animals are evaluated daily beginning 2 days prior totransplant surgery and continuing for the duration of the animal's participation in theexperiment. Transplant heart palpationa) Palpate and score the quality of the heart beat. Record with observations.Heartbeat is graded as A (strong heartbeat), B (weak heartbeat), or C (noheartbeat felt).b) If the heart is no longer beating before the animal reached day 7, the dataobtained 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 placedin separate containers of formalin. Notify Dr. Kerjner when she is available Hind limp paralysisa) If one leg is paralyzed, record observations daily and euthanize if paralysispersists longer than 1 week.b) If both legs are paralyzed, euthanize animals immediately. Notify Dr. Kerjnerand collect native and transplanted hearts in formalin Weigh animals at each evaluation and record with observations.184Appendix II  Animals are kept for 4, 7 or 30 days post surgery and treated daily withimmunosuppressive drugs for 14 to 30 days. Make sure to record injections and anytreatments or manipulations in daily record sheets.This scoring/evaluation system was taken and adapted from the Animal Care and UseGuidelines of the University of Florida.185Appendix IIIAppendix III: List of Publications, Abstract, OralPresentation and AwardsPublished Refereed Papers1. Hunter AL, Kerjner A, Mueller KJ, McManus BM and Granville DJ. (2008).Cytochrome p450 2C enzymes contribute to peri-transplant ischemic injury and cardiacallograft vasculopathy. Am J Transplant. (In revision with invitation to resubmit, January2008)2. Elmi S, Sallam NA, Rahman MM, Teng X, Hunter AL, Moien-Afshari F, Khazaei M,Granville DJ, Laher I. (2008) Sulfaphenazole treatment restores endothelium-dependentvasodilation in diabetic mice. Vascul Pharmacol. 48(1):1-8.3. 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 withcaspase recruitment domain (ARC). FEBS Lett. 581(5):879-84.4. Hunter AL*, Bai N*, Laher I, Granville DJ. (2005). Cytochrome p450 2C inhibitionreduces post-ischemic vascular dysfunction. Vascul Pharmacol. 43: 213-219.5. 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. 86:2969-776. Hunter AL*, Cruz RP*, Cheyne BM, McManus BM, Granville DJ. Cytochrome p450Enzymes and Cardiovascular Disease. (2004). Can J of Physiol and Pharmacol. 82:1053-607. Choy JC, Hung VHY, Hunter AL, Cheung PK, Luo Z, Motyka B, Goping IS, SawchuckT, Bleackley RC, Podor, TJ, McManus BM, and Granville DJ. (2004). Granzyme Binduces smooth muscle cell death in the absence of perforin: implications for theproteolysis of extracellular proteins. Arteriosd Thromb Vasc Biol 24(12):2245-50.8. Upton C, Slack S, Hunter AL, Ehlers A, Roper RL. (2003). Poxvirus orthologousclusters: toward defining the minimum essential poxvirus genome. J Virol. 77:7590-600.Book Chapters1. Hunter AL*, Choy JC*, Granville DJ. (2005). Detection of apoptosis in cardiovasculardiseases, in Molecular Cardiology: Methods and Protocols (Sun Z. ed.). Humana, Totowa,NJ. Vol. 112:277-89186Appendix IIIPublished Abstracts1. Hunter AL, Kerjner A, Mueller KJ, McManus BM, Granville DJ (2007) Cytochromep450 (CYP) 2C Contributes to Cardiac Allograft Vasculopathy. ISHLT 27th AnnualMeeting and Scientific Sessions, April 25 — 28, in San Francisco.2. Hunter AL, Chehal M, McManus BM, Granville DJ (2006) Cytochrome p450 2CIncreases endothelial dysfunction following ischemia and reperfusion. The 3 rd AnnualNational Research Forum for Young Investigators in Cardiovascular and RespiratoryHealth, Winnipeg, MB. Exp Clin Cardiol. 11:1, 50.3. Hunter AL, Bai N, McManus BM, Laher I, Granville DJ. (2005) Inhibition ofCytochrome p4-50 2C Restores Vascular Function Following Global Ischemia andReperfusion. Canadian Cardiovascular Congress 2005, Montreal QC. Canadian Journalof Cardiology Vol 21(C).4. Hunter AL, Bai N, McManus BM, Laher I, Granville DJ. (2005). Sulfaphenazolereduces superoxide generation and improves vascular function following ischemia andreperfusion. Experimental Biology 2005 meeting in San Diego, CA, April 4, 2005. TheFASEB Journal. 19(4): A485.5. Hunter AL Bai N, McManus BM, Laher I, Granville DJ. (2005) Cytochrome p450enzymes contribute to superoxide production and vascular dysfunction followingischemia and reperfusion. The 2nd Annual National Research Forum for YoungInvestigators 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, GranvilleDJ. (2004). Role of Apoptosis Repressor with Caspase Recruitment Domain (ARC) inEndothelial Cell Death. The 1 st Annual National Research Forum for Young Investigatorsin Cardiovascular and Respiratory Health, Winnipeg, MB. Exp Clin Cardiol. 9:56.7. Hunter AL, Chen YL, Chen SC, Gustafsson AB, Gottlieb RA, McManus BM, GranvilleDJ. (2004). TAT-Mediated Protein Transduction of Apoptosis Repressor with CaspaseRecruitment Domain (ARC) in Endothelial and Smooth Muscle Cells. The 93rd AnnualUnited States and Canadian Academy of Pathology Meeting. Vancouver, BC. ModernPathology. 17(Supp 1): 57A8. Granville DJ, Choy JC, Hunter AL, Kerjner A, Goping IS, Sawchick T, Jirik FR,Bleackley C, McManus BM. (2003). Granzyme B-mediated smooth muscle cellapoptosis contributes to medial degeneration in cardiac allograft vasculopathy. AmericanHeart Assoc Scientific Conference on Molecular Mechanisms of Growth, Death andRegeneration in the Myocardium, Snowbird, UT.9. Roper RL, Li G, Chen N, Hunter AL, Buller RML, and Upton C. (2003). CompleteRabbitpox Virus Genome Sequence, Phylogeny and Virulence Factors. 22nd AnnualAmerican Society for Virology Meeting, Davis, California.187Appendix IIIOral PresentationsInvited presentations1. "Cytochrome p450 2C Contributes to Ischemia and Reperfusion Injury and CardiacAllograft Vasculopathy" (2007). Center for Cardiovascular Biology and RegenerativeMedicine, University of Washington Medicine, Department of Pathology, Seattle, WA.2. "Cytochrome p450 2C Contributes to Post-Ischemic Vascular Dysfunction and CardiacAllograft Vasculopathy" (2006). Heart Transplant Research Group at the Alberta StolleryChildren's Hospital, Edmonton, AB.Podium presentations1. "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 CardiacAllograft 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 GlobalIschemia and Reperfusion". (2005). Canadian Cardiovascular Congress 2005, MontrealQC.(l st prize oral presentation from Canadian Society for Atherosclerosis, Thrombosisand Vascular Biology, $500.)4. "Cytochrome p450 2C9 Inhibition Reduces Post-ischemic Superoxide and VascularDysfunction." (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 TransplantationResearch Day, Vancouver BC. (1st prize oral presentation, $500).Awards1. Heart and Stroke Foundation of Canada, Doctoral Research Award, $62,000, 06/06-06/092. Michael Smith Foundation (MSFHR), Junior Trainee Award, $45,000, 09/04-09/063. MSFHR, Senior Trainee Award, $45,000 (partially declined), 06/04-08/084. Canadian Society for Atherosclerosis, Thrombosis and Vascular Biology (CSATVB),2005 Top Oral Presentation Award, $5005. Canadian Cardiovascular Society, 'Have a Heart Bursary', approx $2,500, 10/056. CSATVB, Travel Award, $1,500, 05/057. Canadian Society of Transplantation, Travel Award, $1,500, 05/048. Centre for Blood Research/IMPACT, Best Oral Presentation Award, $150, 20069. UBC, Department of Pathology, 1 st Place Oral Award, $300,06/0510. iCAPTURE Centre, Rookie of the Year Award, $100, 200311. BC Transplant Society, 1st Place Oral Award, $500, 2003188

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