INVESTIGATIONS INTO THE ROLE OF NITRIC OXIDE IN CARDIOVASCULAR DEVXLOPMENT DISEASE: INSIGHTS GAINED FROM GENETICALLY ENGINEERED MOUSE MODELS OF HUMAN DISEASE Tony Lee A thesis submitted in conformity with the requirements for the degree of Master's of Science Institute of Medicai Science University of Toronto @ Copyright by Tony C. Lee (2000) National Library Bibliothèque nationale I*I of Canada du Canada Acquisitions and Acquisitions et Bibliogaphic Services services bibliogaphiques 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON KIA ON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microfonn, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or othewise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. INVESTIGATIONS INTO THE ROLE OF MTNC OXIDE IN CARDIOVASCULAR DEVELOPMENT AND DISEASE: INSIGHTS GAiNED FROM GENETICALLY ENGINEERED MOUSE MODELS OF HUMAN DISEASE B y Tony Lee A thesis submitted in conformity with the requirements for the degree of Master's of Science (2000) Instinite of Medical Science, University of Toronto In the first expenmental series, the antiatherosclerotic effects of Enalûpril vrrsus Irbesartan were compared in the LDL-R knockout mouse model. Both dmgs produced similar reductions in atheroscIerosis, independent of blood pressure, likely due to an increase in biologically available NO levels. In the second sxperimental senes. the contributions of eNOS and iNOS in atherosclerosis and that of eNOS in hem development were evaluated by high cholesterol feeding and seriai examination of hearts from mice deficient in the respective genes. Atherosclerotic lesions were not observed in wild type, eNOS knockout or NOS knockout mice. Interestingly, of the 12 eNOS knockout mice exarnined, 5 were found to have bicuspid aortic valve (BAV), whereas none of the 26 wild type mice exhibited identifiable valvular abnormalities. These results show a strong association between eNOS deficiency and the presence of BAV and provide the first mechanistic insight into the development of this congentid cardiac abnormality. t would like to thank my thesis supervisor, Dr. Duncan Stewart for giving me the opportunity io work in his laboratory and for the guidance and direction he has given me over the years. His passion and cornrnittment to science and medicine will forever remain an inspiration to me. 1 would dso like to thank my prograrn advisory committee members, Dr. Myron Cybulsky and Dr. Peter Liu. tor their constant source of stimulation and enthusiasm in my research. as well as for their advice and cntical analysis of my thesis work. To my fnends and col leagues in the Vascular Biology Research Laboratories at St. Michael's Hospital. thanks for making my time at the lab an enjoyable one. Finally, to rny fàrnily and fiiends who believed in me and encouraged me to reach for my goals. you have made this work tmly meaningfd and I dedicate this to you. Abstract Acknowlegements Table of Contents List of Abbreviations List of Tables and Figures 1. Discovery of Endothelium-Derived Relaxing Factor 2. Use of Genetically Engineered Mice to hvestigate Complex Physiological Traits 2.1 Mouse Models of Atherosclerosis 3. The Biological Chemistry of Nitric Oxide Synthesis 3. Gened Characteristics and Chromosomal Localization of the Nitric Oxide Spthases 3.2 Regdation of Nimc Oxide Synthase Expression in the Cardiovascular S ystem 3.21 nNOS 3.22 NOS 3.23 eNOS 3.3 Subcellular Localization of the Nitric Oxide Synthases 3.4 Inhibiton of Nitric Oxide Biosynthesis 3.4 1 Endogenous Inhibitors of NOS Activity 3.5 Measurement of Nitric Oxide. Nitric Oxide Synthase and Nitric Oxide Synthase Activity 3.5 1 Cherniluminescence 3.52 L-citruhe Assay 3 33 Immunohistochemistry 3.54 Nitric Oxide Electrodes 4. Signaling Mechanisms and Biological Fate of Nitric Oxide 5. Biological Roles of Nitric Oxide in the Cardiovascular System 5.1 Control of Vascular Tone 5.2 Proliferation and Migration of Smooth Muscie Cells and Extr;lcellular Matrix Twnover 5.3 Platelet and Leukocyte Adhesion 5.4 Reguiation of Apoptosis 5.5 Insights Gained from Knockout Mice 5.51 nNOS 5.52 eNOS 5.53 NOS 5.6 Cardiovascular Development 6. Endothelid Dyshcnction and the Role of Nitric Oxide in Atherosclerosis 6.1 Pathogenesis of Atherosclerosis 6.2 Impaired Biologicai Activity of Ninic Oxide in Atherosclerotic Vessels 25 6.3 Mechanisrns and Functional Consequences of Impaired Nitric Oxide Activity 26 6.3 1 Altered Production and tncreased Oxidative Inactivation by NO 26 6.32 Functional Antagonism by Endothelium-Derived Vasoconstrictors 28 6.33 Irnpaired Endothelial Signai Transduction 29 7. Nitric Oxide and the Renin-Angiotensin System in Atherosclerosis 29 HYPOTHESISAND OEMECTIVES 32 (CHAPTERONE) Differential E ffects of Renin- Angiotensin System Blockade on the Initiation and 32 Progression of Atherosclerosis in the LDL Receptor Knockout Mouse (CHAPTERTWO) Cardiac Development and Atherosclerosis in Mice Lacking Specific Isoforms of 33 Nitric Oxide Synthase 1. Pilot Blood Pressure Study 35 a) Animais b) Subcutaneous Drug Administration C) Blood Pressure Measurement d) Pressor Response to Angiotensin I e) Expenmental Protocol Effect of RAS Blockade on Atherosclerosis a) Animais b) Sample Size Calculation c) Expenmental Protocol d) Pressor Response to Angiotensin 1 e) Body Weight and Plasma Cholesterol Determination t') Tissue and Slide Preparation g) Oil Red O Lipid Staining and Quantification of Aonic Atherosclerotic Lesions h) Statistical Analysis 1. Pi10 t Blood Pressure Study 42 a) Blood Pressure Response to infusion of Angiotensin I in 42 Wild Type Mice b) Blood Pressure Response to Angiotensin I Following RAS Blockade 45 2. Effect of RAS Blockade on Atherosclerosis 46 a) Blood Pressure Response to Ang I Post Treatment 46 b) Body Weight and Plasma Cholesterol Levels 47 C) Oil Red O Lipid Staining and Quantification of Atherosclerotic Plaques 49 Development of Atherosclerosis in Mice Lackiag the Endothelial or 55 Inducible Isoforms of Nitric Oxide Synthase a) Mice b) Tissue Preparation and Histology Abnormal Aortic Valve Development in Mice Lacking eNOS a) Mice b) Blood Pressure Measurements C) Histological Analysis d) immunohistochemistry e) Vascular Casting f) Statistical Analysis vii 1. Development of Atherosclerosis in Mice Lacking the Endothelial or 59 Inducible Isoforms of Nitric Oxide Synthase .3 Abnormal Aortic Valve Development in Mice Lacking eNOS a) Blood Pressure Measurements b) Histological and Imrnunohistochemical Analysis C) Vascular Casting 1. Development of Atherosclerosis in Mice Lacking the Endothebal or 66 Inducible Isoforms of Nitric Oxide Synthase 2. Abnormal Aortic Valve Development in Mice Lacking eNOS 67 ACh acetylcholine ACE angiotensin converting enzyme ADMA asy nunetriz Jiiwthy iarginim A% 1 angiotensin 1 hgII angiotensin II Apo-E apolipoprotein E ATi angiotensin receptor subtype 1 AT2 angiotensin receptor subtype II BAV bicuspid aortic valve Brr, tetrahydrobiopterin CaM calmodulin cGMP guanosine 3 3'-cyclic monophosphate EDRF endothelium-derived relaxing factor eNOS endothelial nimc oxide synthase GTP guanosine triphosphate HMG-COA 3 -hydroxy-3 -methylglutaryl-coenzyme A ICAM-I intercellular adhesion molecule I 1NOS inducible nitric oxide synthase LDL-R low density lipoprotein receptor LPC lysophosphatidylcholine LPS Iipopolysaccharide MCP-1 monocyte chemoattractant protein 1 MMP matrix rnetalloproteinase NADPH nicotinarnide adenine dinucleo tide phosphate nNOS neuronal nitric oxide synthase NO nitric oxide NO' nitrosoniurn ion NO; nitrite NO3' nitrate NOS nitric oxide synthase OÎ' superoxide 03 ozone ONOO' peroxynitrite oxLDL oxidized low density lipoprotein RAS renin-angiotensin sy stem sGC soluble guanylate cyclase SOD superoxide dismutase SSRE shear stress response element TNF-a tumor necrosis factor alpha TNF-P tumor necrosis factor beta VCAM-1 vascular ce11 adhesion rnolecule 1 VEGF vascular endothelid growth factor LISTOF TABLESAND FIGURES Item Title Page Table 1 Aortic lumen diameters taken îiom vascular casts Figure 1 Experimental protocol for pilot study to evaluate the abilities of Enalapril and Irbesartan to block the pressor effect of acute hg1 infusion Figure 2 Expenmental protocol for the Atherosclerosis Drug Study Figure 3 Sarnple trace of the blood pressure response of a C57BU6J wild- type animal to the infusion of hgI Figure 4 Log dose-response relationship for the effect of increasing concentrations of hg1 on % increase in MAP Figure 5 Blood pressure response to Ang 1 infusion following 7-day RAS blockade in C57BU61 wild type mice Figure 6 Effect of Ang I on MAP in LDL-R knockout mice following 12 weeks of hi& cholesterol diet and RAS blockade Figure 7 Body weights of LDL-R knockout mice following 12 weeks of high cholesterol diet and treatment with Enalapril or Irbesartan Figure 8 Plasma cholesterol levels of LDL-R knockout mice following 12 weeks of