Zou Wei. / Thesis submitted in: December 2001. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 98-119). / Abstracts in English and Chinese. / Declaration --- p.i / Acknowledgements --- p.ii / Publication lists --- p.iii / Abstract --- p.ix / Abbreviations --- p.xiii / List of tables and figures --- p.xiv / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1. --- Endothelium-dependent relaxation in coronary and pulmonary circulation --- p.1 / Chapter 1.1.1. --- Endothelium-derived relaxing factors --- p.2 / Chapter 1.1.1.1. --- Nitric Oxide --- p.3 / Chapter 1.1.1.2. --- PGI2 --- p.5 / Chapter 1.1.1.3. --- EDHF --- p.6 / Chapter 1.1.2. --- EDHF in coronary and pulmonary circulation --- p.8 / Chapter 1.1.2.1. --- EDHF in coronary circulation --- p.8 / Chapter 1.1.2.2. --- EDHF in pulmonary circulation --- p.9 / Chapter 1.2. --- Effect of hyperkalemia on EDHF-mediated relaxation --- p.10 / Chapter 1.3. --- Organ Preservation Solutions --- p.13 / Chapter 1.3.1. --- Euro-Collins solution --- p.14 / Chapter 1.3.2. --- University of Wisconsin solution --- p.15 / Chapter Chapter 2: --- Objectives and research approaches --- p.16 / Chapter 2.1. --- Objectives --- p.16 / Chapter 2.1.1. --- "Endothelium-dependent relaxation resistant to INDO, L-NNA, and HbO in porcine and pulmonary coronary micro-arteries" --- p.16 / Chapter 2.1.2. --- "EET11,12 and EDHF-mediated function in porcine coronary micro-arteries" --- p.17 / Chapter 2.1.3. --- "Comparison of EC or UW solution on endothelium-dependent relaxation resistant to INDO, l-NNA, and HbO in porcine pulmonary arteries" --- p.17 / Chapter 2.2. --- Research approaches --- p.18 / Chapter 2.2.1. --- "Endothelium-dependence of the relaxation by BK or EET11,12" --- p.18 / Chapter 2.2.2. --- Effect of hypothermic storage with EC and UW solution on EDHF-related relaxation --- p.18 / Chapter 2.2.3. --- Time-dependent alteration of endothelium-dependent relaxation in pulmonary micro-arteries by EC and UW solution --- p.19 / Chapter 2.2.4. --- Effect of HbO in endothelium-dependent relaxation --- p.19 / Chapter Chapter 3: --- Material and Methods --- p.21 / Chapter 3.1. --- General Methods --- p.21 / Chapter 3.1.1. --- Porcine heart and lung collection and transportion / Chapter 3.1.2. --- Myograph --- p.21 / Chapter 3.1.3. --- Myosight --- p.24 / Chapter 3.1.4. --- Anatomizing blood vessel --- p.24 / Chapter 3.1.5. --- Mounting --- p.24 / Chapter 3.1.6 --- Normalization --- p.26 / Chapter 3.1.6.1. --- Normalization of coronary micro-artery --- p.27 / Chapter 3.1.6.2. --- Normalization of pulmonary micro-artery --- p.28 / Chapter 3.1.7. --- Precontraction --- p.30 / Chapter 3.1.8. --- Endothelium-dependent relaxation --- p.31 / Chapter 3.2. --- Coronary artery studies --- p.32 / Chapter 3.2.1. --- Porcine heart harvest and anatomy --- p.32 / Chapter 3.2.2. --- Characteristic of histology of porcine coronary micro-artery --- p.32 / Chapter 3.3. --- Pulmonary artery studies --- p.35 / Chapter 3.3.1. --- Porcine lung harvest and anatomy --- p.35 / Chapter 3.3.2. --- Characteristic of histology of porcine pulmonary micro- artery --- p.36 / Chapter 3.4. --- Drugs --- p.41 / Chapter 3.4.1. --- Drugs --- p.41 / Chapter 3.4.2. --- Preparation of oxyhemoglobin solution --- p.41 / Chapter 3.5. --- Statistical Analysis --- p.42 / Chapter 3.5.1. --- Calculation of EC50 --- p.42 / Chapter 3.5.2. --- Statistical analysis --- p.42 / Chapter Chapter 4: --- "Epoxyeicosatrienoic Acids (EET11,12) May Partially Restore EDHF-Mediated Function in Coronary Micro-Arteries" --- p.43 / Chapter 4.1. --- Abstract --- p.43 / Chapter 4.2. --- Introduction --- p.44 / Chapter 4.3. --- Experimental Protocol --- p.45 / Chapter 4.3.1. --- Precontraction --- p.45 / Chapter 4.3.2. --- "EDHF-mediated (INDO, L-NNA, and HbO-resistant) relaxation" --- p.45 / Chapter 4.3.3. --- "EET11,12-mediated relaxation after exposure to hyperkalemia" --- p.46 / Chapter 4.3.4. --- "The effect of incubation with EET11,12 on the BK-induced, EDHF-mediated relaxation" --- p.46 / Chapter 4.4. --- Results --- p.47 / Chapter 4.4.1. --- Resting force --- p.47 / Chapter 4.4.2. --- HbO and U46619-induced contraction force --- p.48 / Chapter 4.4.3. --- "EET11,12-induced relaxation in coronary micro-arteries after exposure to hyperkalemia" --- p.49 / Chapter 4.4.4. --- "The EDHF-mediated relaxation to BK resistant to INDO, l- NNA,and HbO" --- p.51 / Chapter 4.4.4.1. --- Incubated in either hyperkalemic solution (K+ 20mmol/L) or Krebs' solution (control) --- p.51 / Chapter 4.4.4.2. --- "Incubated in either hyperkalemic solution (K+ 20mmol/L) plus EET11,12 or Krebs' solution (control)" --- p.53 / Chapter 4.5. --- Discussion --- p.57 / Chapter 4.5.1. --- EDHF plays an important role in the coronary micro-arteries --- p.57 / Chapter 4.5.2. --- "EDHF-mediated (INDO, l-NNA, and HbO-resistant) relaxation in the coronary micro-arteries" --- p.58 / Chapter 4.5.3. --- "EET11,12 may partially mimic the EDHF-mediated relaxation in the porcine coronary micro-artery" --- p.59 / Chapter 4.5.4. --- "Effect of EET11,12 added in hyperkalemia may partially restore the EDHF-mediated relaxation in the porcine coronary micro-arteries" --- p.59 / Chapter Chapter 5: --- Impaired EDHF-Mediated Relaxationin Porcine Pulmonary Micro-arteries by Cold Store with Euro-Collin's and University of Wisconsin Solution --- p.61 / Chapter 5.1. --- Abstract --- p.61 / Chapter 5.2. --- Introduction --- p.62 / Chapter 5.3. --- Experimental Protocol --- p.64 / Chapter 5.3.1. --- Precontraction --- p.64 / Chapter 5.3.2. --- "Role of EDHF-mediated (INDO, L-NNA and HbO-resistant) relaxation in porcine pulmonary micro-arteries by BK orA23187" --- p.64 / Chapter 5.3.3. --- Effect of hyperkalemia or preservation solutions (EC or UW) on the EDHF-mediated relaxation by BK or A23187 --- p.65 / Chapter 5.3.3.1. --- The effect of hyperkalemia --- p.65 / Chapter 5.3.3.2. --- Effect of EC solution on the EDHF-mediated relaxation --- p.65 / Chapter 5.3.3.3. --- Effect of UW solution on the EDHF-mediated relaxation --- p.66 / Chapter 5.3.3.4. --- The effect of UW and EC solutions on the contractility of the pulmonary micro-artery --- p.66 / Chapter 5.4. --- Results --- p.66 / Chapter 5.4.1. --- Resting force --- p.66 / Chapter 5.4.2. --- U46619-induced contraction force --- p.67 / Chapter 5.4.3. --- Role of EDHF-mediated relaxation induced by BK or A23187 --- p.67 / Chapter 5.4.4. --- The effect of hyperkalemia --- p.71 / Chapter 5.4.5. --- Effect of EC solution on the EDHF-mediated relaxation --- p.72 / Chapter 5.4.6. --- Effect of UW solution on the EDHF-mediated relaxation --- p.73 / Chapter 5.4.7. --- The effect of UW and EC solution on the contractility of the pulmonary micro-artery --- p.73 / Chapter 5.5. --- Discussion --- p.77 / Chapter 5.5.1. --- EDHF-mediated endothelial function exists in the pulmonary micro-circulation --- p.77 / Chapter 5.5.2. --- Hyperkalemia exposure reduces EDHF-related relaxation and possible mechanism --- p.78 / Chapter 5.5.3. --- The effect of EC and UW solutions on the EDHF-media relaxation in the pulmonary micro-arteries --- p.79 / Chapter Chapter 6: --- General Discussion --- p.82 / Chapter 6.1. --- Endothelium-dependent vasodilators: BK and A23187 --- p.82 / Chapter 6.2. --- EDHF in porcine coronary and pulmonary micro-arteries --- p.84 / Chapter 6.2.1. --- EDHF in porcine coronary micro-arteries --- p.84 / Chapter 6.2.2. --- EDHF in porcine pulmonary micro-arteries --- p.87 / Chapter 6.2.3. --- Vascular stretch and release of endothelium-derived vasodilators --- p.87 / Chapter 6.2.4. --- "EET11,12" --- p.88 / Chapter 6.3. --- "Endothelium-dependent relaxation resistant to INDO, L- NNA, and HbO in porcine coronary and pulmonary microcirculation" --- p.89 / Chapter 6.4. --- "Alteration of endothelium-dependent relaxation resistant to INDO, l-NNA, and HbO after exposure to hyperkalemia" --- p.90 / Chapter 6.5. --- "Alteration of endothelium-dependent contraction resistant to INDO, L-NNA, and HbO after exposure to EC or UW solutions" --- p.91 / Chapter 6.6. --- Clinical implications --- p.92 / Chapter 6.7. --- Limitations --- p.93 / Chapter 6.7.1. --- Common limitations --- p.93 / Chapter 6.7.2. --- Limitation of in vitro study --- p.93 / Chapter 6.8. --- Future work --- p.94 / Chapter Chapter 7: --- Conclusion --- p.96 / References --- p.98 / Appendies / "Wei Zou, Qin Yang, Anthony PC Yim, & Guo-Wei He Epoxyeicosatrienoic acids (EET11,12) may partially restore endothelium- derived hyperpolarizing factor-mediated function in coronary micro- arteries. Annals of Thoracic Surgery. 2001; 72(12): 1970~1976."

Yau Ho Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 131-144). / Abstracts in English and Chinese. / DECLARATION --- p.I / ACKNOWLEDGEMENTS --- p.II / ENGLISH ABSTRACT --- p.III / CHINESE ABSTRACT --- p.VI / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Oxidative Stress --- p.1 / Chapter 1.1.1 --- Historical Background of reactive oxygen/nitrogen species --- p.1 / Chapter 1.1.2 --- What is Oxidative Stress? --- p.3 / Chapter 1.1.3 --- Reactive Oxygen Species (ROS) --- p.4 / Chapter 1.1.3.1 --- Superoxide anion (02-) --- p.4 / Chapter 1.1.3.2 --- Hydrogen peroxide (H202) --- p.5 / Chapter 1.1.3.3 --- Hydroxyl radical --- p.6 / Chapter 1.1.3.4 --- Nitric oxide (NO) --- p.7 / Chapter 1.2 --- Cardiovascular System --- p.8 / Chapter 1.2.1 --- Enzymatic and Non-enzymatic Sources of ROS in Cardiovascular System --- p.8 / Chapter 1.2.1.1 --- NADPH oxidase --- p.8 / Chapter 1.2.1.2 --- Hypoxanthine-Xanthine oxidase (HX-XO) --- p.9 / Chapter 1.2.1.3 --- Nitric oxide synthase (NOS) --- p.10 / Chapter 1.2.1.4 --- Mitochondrial electron transport chain (ETC) --- p.11 / Chapter 1.2.1.5 --- Cyclooxygenase --- p.11 / Chapter 1.2.1.6 --- Lipoxygenae --- p.12 / Chapter 1.2.1.7 --- Endoplasmic reticulum --- p.12 / Chapter 1.2.2 --- ROS/RNS Scavenging Systems --- p.13 / Chapter 1.2.2.1 --- Superoxide dismutase (SOD) --- p.13 / Chapter 1.2.2.2 --- Catalase --- p.14 / Chapter 1.2.2.3 --- Glutathione peroxidase --- p.15 / Chapter 1.2.2.4 --- Non-enzymatic antioxidants --- p.15 / Chapter 1.2.3 --- Factors that stimulate ROS production in cardiovascular system --- p.18 / Chapter 1.2.3.1 --- Oxygen tension --- p.18 / Chapter 1.2.3.2 --- "Flow, Shear, and Stretch as an initial stimulus for endothelial oxidant signalling" --- p.18 / Chapter 1.2.3.3 --- Activation of rennin-angiotensin system promote oxidative stress in cardiovascular system --- p.19 / Chapter 1.2.3.4 --- Regulation of vascular ROS production by vasoactive substances --- p.19 / Chapter 1.2.4 --- Regulation of vascular tone in Cardiovascular System by ROS/RNS --- p.20 / Chapter 1.2.4.1 --- Regulation of vascular tone --- p.20 / Chapter 1.2.5 --- Pathophysiological Effects of ROS --- p.23 / Chapter 1.2.5.1 --- Cellular injury by lipid peroxidation --- p.23 / Chapter 1.2.5.2 --- Role of ROS in immune defence --- p.23 / Chapter 1.2.5.3 --- Redox regulation of cell adhesion --- p.24 / Chapter 1.2.6 --- Evidences from Clinical Studies of Oxidative Stress-Related Vascular Diseases --- p.25 / Chapter 1.2.6.1 --- Hyperlipidaemia --- p.25 / Chapter 1.2.6.2 --- Hypertension --- p.25 / Chapter 1.2.6.3 --- Chronic heart failure (CHF) --- p.26 / Chapter 1.2.6.4 --- Chronic renal failure (CRF) --- p.26 / Chapter 1.2.6.5 --- Atherosclerosis --- p.27 / Chapter 1.2.6.6 --- Ischemia/reperfusion (I/R) injury --- p.27 / Chapter 1.2.7 --- Role of Vascular Endothelium in Oxidative Stress --- p.29 / Chapter 1.2.8 --- Role of Ca in oxidative stress in cardiovascular system --- p.29 / Chapter 1.2.8.1 --- Calcium Signaling in Vascular Endothelial Cells --- p.30 / Chapter 1.2.9 --- ROS effect on endothelial Ca2+ --- p.31 / Chapter 1.2.9.1 --- Multiple targets of ROS on intracellular Ca2+ mobilization --- p.32 / Chapter 1.2.9.2 --- Reports of H202-induced Ca2+ release in various cell types --- p.33 / Chapter 1.2.9.3 --- Reported effects of H202 on agonist-induced Ca2+ signal --- p.34 / Chapter 1.2.9.4 --- Differences between macrovessels and microvessels --- p.34 / Chapter 1.3 --- TRP Channel --- p.41 / Chapter 1.3.1 --- Discovery of Drosophila TRP --- p.41 / Chapter 1.3.2 --- Mammalian TRP subfamily --- p.41 / Chapter 1.3.3 --- General topology of TRP channel --- p.42 / Chapter 1.3.4 --- Interactions of oxidative stress with TRP channels --- p.44 / Chapter 1.3.5 --- The role of TRPC3 and TRPC4 in oxidative stress --- p.44 / Chapter 1.3.6 --- TRPM subfamily --- p.44 / Chapter 1.3.6.1 --- Expression of TRPM2 --- p.45 / Chapter 1.3.6.2 --- Dual Role of TRPM´2ؤChannel and Enzyme --- p.45 / Chapter 1.3.6.3 --- Regulatory mechanisms of TRPM2 --- p.46 / Chapter 1.3.6.3.1 --- ADP-ribose (ADPR) directly regulating --- p.46 / Chapter 1.3.6.3.2 --- NAD regulating --- p.46 / Chapter 1.3.6.3.3 --- Oxidative stress regulating independent of ADPR or NAD --- p.47 / Chapter 1.4 --- Cell Death Induced by Oxidative Stress --- p.48 / Chapter 1.4.1 --- Redox status as a factor to determine cell death --- p.48 / Chapter 1.4.2 --- Role of TRPM2 in oxidative stress-induced cell death --- p.48 / Chapter 1.5 --- Aims of the Study --- p.49 / Chapter Chapter 2: --- Materials and Methods --- p.50 / Chapter 2.1 --- Functional Characterization of TRPM2 by Antisense Technique --- p.50 / Chapter 2.1.1 --- Restriction Enzyme Digestion --- p.50 / Chapter 2.1.2 --- Purification of Released Inserts and Cut pcDNA3 Vectors --- p.51 / Chapter 2.1.3 --- "Ligation of TRPM2 Genes into Mammalian Vector, pcDNA3" --- p.52 / Chapter 2.1.4 --- Transformation for the Desired Clones --- p.52 / Chapter 2.1.5 --- Plasmid DNA Preparation for Transfection --- p.53 / Chapter 2.1.6 --- Confirmation of the Clones --- p.53 / Chapter 2.1.6.1 --- Restriction Enzymes Strategy --- p.53 / Chapter 2.1.6.2 --- Polymerase Chain Reaction (PCR) Check --- p.54 / Chapter 2.1.6.3 --- Automated Sequencing --- p.55 / Chapter 2.2 --- Establishing Stable Cell Lines --- p.56 / Chapter 2.2.1 --- Cell Culture --- p.56 / Chapter 2.2.2 --- Geneticin Selection --- p.57 / Chapter 2.3 --- Expression of TRPM2 in Transfected and non-Transfected H5V Cells --- p.57 / Chapter 2.3.1 --- Protein Sample Preparation --- p.57 / Chapter 2.3.2 --- Western Blot Analysis --- p.58 / Chapter 2.3.3 --- Protein Expression Analysis --- p.59 / Chapter 2.4 --- "Immunolocalization of TRPM2 in Human Heart, Cerebral Artery, Renal, Hippocampus and Liver" --- p.59 / Chapter 2.4.1 --- Paraffin Section Preparation --- p.59 / Chapter 2.4.2 --- Immunohistochemistry --- p.60 / Chapter 2.5 --- [Ca2+ ］i Measurement in Confocal Microscopy --- p.62 / Chapter 2.5.1 --- Cytosolic Ca2+ measurement --- p.62 / Chapter 2.5.2 --- Measuring the Ca2+ in the Internal Calcium Stores --- p.63 / Chapter 2.5.3 --- Data Analysis --- p.64 / Chapter 2.6 --- Examining Cell Death Induced by H2O2 by DAPI Staining --- p.65 / Chapter 2.6.1 --- DAPI Staining --- p.65 / Chapter Chapter 3: --- Results --- p.66 / Chapter 3.1 --- Superoxide Anion-Induced [Ca 2+]i rise in H5V Mouse Heart Microvessel Endothelial Cells --- p.66 / Chapter 3.1.1 --- Superoxide Anion-induced [Ca2+ ]i Rise --- p.66 / Chapter 3.1.2 --- Effect of Catalase on the Superoxide Anion-induced [Ca2+]i］］ Rise --- p.66 / Chapter 3.1.3 --- IP3R inhibitor Inhibits Superoxide anion-induced [Ca 2+]i Rise --- p.67 / Chapter 3.1.4 --- Effect of Phospholipase A2 Inhibitor on Superoxide anion- induced [Ca2+]i Rise --- p.67 / Chapter 3.1.5 --- Effect of Hydroxyl Radical Scavenger on Superoxide Anion- induced [Ca2+]i Rise --- p.68 / Chapter 3.2 --- Hydrogen Peroxide-induced Ca2+ Entry in Mouse Heart Microvessel Endothelial Cells --- p.74 / Chapter 3.2.1 --- Hydrogen Peroxide Induces [Ca2 +]i rise in H5V Mouse Heart Microvessel Endothelial Cells --- p.74 / Chapter 3.2.2 --- Hydrogen Peroxide Induces [Ca 2+]i rise in two phases (Rapid and Slow response) --- p.74 / Chapter 3.2.3 --- Hydrogen Peroxide Induces [Ca 2+]i rise in a Extracellular Ca + Concentration Dependent Manner --- p.77 / Chapter 3.3 --- Hydrogen Peroxide Reduces Agonist-induced [Ca2+]i rise --- p.79 / Chapter 3.3.1 --- Hydrogen Peroxide Reduces ATP-induced [Ca2+ ]i rise in a H2O2 Concentration Dependent Manner --- p.79 / Chapter 3.3.2 --- Hydrogen Peroxide Reduces ATP-induced [Ca 2+]i rise in a H2O2 Incubation Time Dependent Manner --- p.79 / Chapter 3.3.3 --- Hydrogen Peroxide Reduces the ATP-induced Intracellular Ca2+ Release --- p.80 / Chapter 3.3.4 --- XeC Inhibited H202-induced [Ca2+]i rise --- p.80 / Chapter 3.3.5 --- Hydrogen Peroxide Partially Depletes Internal Ca2+ Stores --- p.81 / Chapter 3.4 --- Dissecting Signal Transduction Pathways in H202-induced [Ca2+]i rise --- p.82 / Chapter 3.4.1 --- Effect of Phospholipase C Inhibitor on H202-induced [Ca2 +]i rise --- p.82 / Chapter 3.4.2 --- Effect of Phospholipase A2 Inhibitor on H202-induced [Ca 2+]i rise --- p.83 / Chapter 3.4.3 --- Effect of hydroxyl radical scavenger on H2O2-induced [Ca 2+]i rise --- p.83 / Chapter 3.5 --- Functional Role of TRPM2 Channel in H202-induced [Ca2+]i Rise in H5V Cells --- p.92 / Chapter 3.5.1 --- Expression of TRPM2 and the Effect of TRPM2 Antisense Construct on TRPM2 Protein Expression --- p.92 / Chapter 3.5.2 --- Effect of Antisense TRPM2 on H202-induced Ca2+ Entry --- p.94 / Chapter 3.6 --- H202-induced Cell Death --- p.101 / Chapter 3.7 --- Expression Pattern of TRPM2 Channel in Vascular System --- p.104 / Chapter 3.7.1 --- Immunolocalization of TRPM2 in Human Cerebral Arteries --- p.104 / Chapter 3.7.2 --- Immunolocalization of TRPM2 in Human Cardiac Muscles --- p.105 / Chapter 3.7.3 --- Immunolocalization of TRPM2 in Human Kidney --- p.105 / Chapter Chapter 4: --- Discussion --- p.113 / Chapter 4.1 --- Oxidative modification of Ca2+ homeostasis --- p.113 / Chapter 4.2 --- Pathophysiological effects of ROS on endothelium --- p.113 / Chapter 4.3 --- Effects of ROS on microvascular endothelial Ca2+ reported by other investigators --- p.115 / Chapter 4.4 --- Studies of the effect of HX-XO on cytosolic [Ca2+]i --- p.116 / Chapter 4.4.1 --- Role of 0´2Ø- and H202 in HX-XO-induced [Ca2+]i elevation --- p.116 / Chapter 4.4.2 --- IP3R involvement in HX-XO-evoked Ca + movements in H5V cells --- p.118 / Chapter 4.4.3 --- PLA2 involvement in HX-XO experiment --- p.119 / Chapter 4.5 --- Studies of the effect of direct H202 application on cytosolic [Ca2+]i --- p.120 / Chapter 4.5.1 --- Hydrogen Peroxide Induced [Ca2 +]i rise in a Extracellular Ca2 + Concentration Dependent Manner --- p.120 / Chapter 4.5.2 --- Hydrogen Peroxide Induced [Ca 2+]i rise in two phases (Rapid and Slow response) --- p.121 / Chapter 4.6 --- Effect of H202 on ATP-induced Ca2+ response --- p.121 / Chapter 4.6.1 --- H202 inhibited ATP-induced Ca2+ release in a concentration and time dependent manner --- p.121 / Chapter 4.6.2 --- IP3R involvement and store depletion in H202 experiment --- p.123 / Chapter 4.7 --- Dissecting Signal Transduction Pathways in H202-induced [Ca2+]i rise --- p.124 / Chapter 4.7.1 --- PLC involvement in H2O2 experiment --- p.124 / Chapter 4.7.2 --- PLA2 involvement in H2O2 experiment --- p.125 / Chapter 4.7.3 --- Hydroxyl radical did not involve in H2O2 experiment --- p.125 / Chapter 4.8 --- Functional Studies of TRPM2 --- p.127 / Chapter 4.8.1 --- Expression of TRPM2 in H5V on protein level --- p.127 / Chapter 4.8.2 --- TRPM2 involvement in the Ca2+ signalling in response to H2O2 in H5V cells --- p.127 / Chapter 4.9 --- H202 concentration in my projec´tؤphysiological or pathological? --- p.128 / Chapter 4.10. --- H20´2ؤTRPM´2ؤCell death --- p.129 / Chapter 4.11 --- Expression of TRPM2 in human blood vessels and other tissues --- p.130 / References --- p.131

Based on the results aforementioned, I went on in the second part of the study to examine the impact of aging on EDCF-mediated contractions - the alterations of COX-2-mediated endothelium-dependent contractions and the associated release of prostaglandin(s) in the aortae from aged (>18 month-old) hamsters. Endothelium-dependent contractions in the presence of NG-nitro-L-arginine methyl ester (L-NAME) were significantly greater in the aortae from aged hamsters and contractions could also be observed without L-NAME, which were sensitive to COX-2 inhibitors and TP receptor antagonists. The levels of COX-2 expression, the release of PGF2alpha and vascular sensitivity to PGF 2alpha were augmented in aortae of aged hamsters. The present results indicate a positive impact of aging on COX-2-derived PGF2alpha-mediated endothelium-dependent contractions. / In the first part of the study, I investigated whether COX-2 participated in the occurrence of endothelium-dependent contractions in the aortae from young (-3 month-old) hamsters and identified the most possible EDCF. Endothelium-dependent contractions were elicited by acetylcholine and abolished by COX-2 inhibitors (NS-398, DuP-697 and celecoxib) and thromboxane-prostanoid (TP) receptor antagonists (S 18886, L-655,240 and GR 32191), but not by COX-1 inhibitors (valeryl salicylate and sc 560). RT-PCR and Western blot analysis using aortae with and without endothelium revealed that the COX-2 expression was localized mainly in the endothelium. Levels of prostangladin F2alpha (PGF2alpha ) and prostacyclin (PGI2) increased in response to acetylcholine and the release of both prostaglandins was inhibited by COX-2 but not COX-1 inhibitors. Exogenous PGF2alpha but not PGI2 caused contractions at a concentration that corresponded to the amount released endogenously. The release of PGF2alpha was not affected by the presence of nitric oxide (NO). The results of the present study suggest that a novel constitutive role of COX-2 in endothelium-dependent contractions, with its metabolites PGF2alpha acting as a physiological EDCF in the young hamster aortae. / In the third part of the study, I investigated the relationship and the intracellular signaling cascades linking two pro-inflammatory factors Ang II and COX-2, and tested whether COX-2 mediated the Ang II-induced vascular pathogenesis. Eight hour-incubation with 100 nmol/L Ang II resulted in maximal COX-2 expression in primary rat endothelial cells and it was inhibited by losartan and RNA synthesis inhibitor (actinomycin-D). Inhibitors of either p38 MAPK or ERK1/2 (respectively SB 202190 and PD 98059) decreased the COX-2 expression, and co-treatment with both inhibitors caused an additive effect, suggesting a joint mediation through both kinases. Protein kinase C (PKC) inhibitor (GF109203X), and particularly, the specific PKCdelta inhibitor (rottlerin), prevented Ang II-induced phosphorylation of ERK1/2 and COX-2 expression, indicating an upstream regulation of ERK1/2 by PKC delta. A pivotal role of PKCdelta in Ang II-induced COX-2 expression was further supported by a similar stimulatory effect of PKC activator, signified by the Ang II-stimulated translocation of PKCdelta to the membrane and confirmed by its phosphorylation (Tyr311). Small interfering RNA targeting PKCdelta (siPKCdelta) diminished COX-2 expression, which was abrogated in siPKCdelta-treated cells treated with SB 202190, confirming the parallel pathways of PKC delta-ERK1/2 and p38 MAPK. Aortae and renal arteries from Ang II-infused rats exhibited an increased endothelial COX-2 expression and impaired acetylcholine-induced relaxation that was normalized by celecoxib. Human mesenteric arteries incubated with Ang II demonstrated elevated endothelial COX-2 and MCP-1 expressions, of which the former was inhibited by SB 202190 plus rottlerin and the latter prevented by COX-2 inhibitor celecoxib. Renal arteries from hypertensive or diabetic patients revealed an exaggerated expression of COX-2 and MCP-1 in the endothelium. The present novel findings indicate that the activation of PKCdelta-ERK1/2 and p38 MAPK is critical in Ang II-induced COX-2 up-regulation in endothelial cells, and identify a COX-2-dependent pro-atherosclerotic cytokine MCP-1. (Abstract shortened by UMI.) / Wong, Siu Ling. / Adviser: Huang Yu. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 192-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Wong, Wing Tak Jack. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 204-227). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (M.Sc.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Experimental Medicine, Department of Medicine. Title from pdf file main screen (viewed on January 17, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 147-164.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references.

Diss. (sammanfattning) Umeå : Univ., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2008" Includes bibliographical references.