活性氧在內皮細胞生理發展比如細胞生長增殖和病理中起到非常重要的作用。在病理條件下，活性氧在血管功能失調和重構起到關鍵作用。氧化應激現在被認為存在於多種形式的心血管疾病中。諸多證據表明著活性氧誘導的心血管系統中很多功能異常之前會伴隨有細胞內鈣離子濃度的上升。 / 在本論文的第一個部分，我比較了活性氧在大血管（主動脈）和小血管（腸系膜動脈）的內皮細胞裡引起的鈣應激的相似和差異之處。在這兩種細胞中，活性氧均可引起細胞內鈣離子濃度的上升。這種鈣離子濃度增加可被磷酸酯酶C (PLC) 的抑製劑U73122或者磷酸肌醇受體 (IP₃R) 抑製劑 （Xestospongin C， XeC）大幅度的減弱。此外，用過氧化氫預處理後的細胞會降低細胞對ATP的鈣應激反應。這種鈣應激反應的抑制可能是由於過氧化氫引發的鈣庫流失。令人關注的是，腸系膜動脈的內皮細胞對過氧化氫的作用更為敏感。次黃嘌呤 (hypoxanthine; HX) 加上黃嘌呤(xanthine; XO) 也能引起這兩種內皮細胞鈣離子濃度的上升，而這種鈣離子的增加源於超氧陰離子而不是氫氧離子。在腸系膜動脈的內皮細胞中，過氧化氫在此事件中起到的作用明顯比在主動脈細胞大。總之，過氧化氫可以引起大血管和小血管的內皮細胞裡磷酸酯酶C-磷酸肌醇受體依賴的鈣應激反應。而這種鈣應激後的鈣庫耗竭會對ATP引起的鈣應激起作用。綜上所述，小血管的內皮細胞的鈣應激比大血管的內皮細胞對過氧化氫更為敏感。 / 基於以上的結果，在第二部分的內容中，我們以培植的微血管內皮細胞系（H5V）為小血管內皮細胞的模型，研究了TRPM2通道在過氧化氫誘導的的鈣應激和凋亡中的作用。TRPM2是表達在動物是血管內皮組織中的氧化敏感的和陽離子無選擇性通道。我們開發了TRPM2通道的抑制性抗體 (TM2E3)，這種抗體可以結合到TRPM2通道的離子孔道的E3區域。對H5V細胞進行TM2E3的預處理後，可以降低細胞對過氧化氫刺激下的鈣離子的增加。用TRPM2特異的短發卡核糖核酸 （shRNA）也有同樣的抑制反應。我們用了3種方法來檢測過氧化氫誘導的細胞凋亡：四甲基偶氮唑盐（MTT）檢測，脫氧核糖核酸凋亡片段的檢測和4,6-联脒-2-苯基吲哚(DAPI) 核染色。基於以上的試驗結果，TM2E3 和TRPM2特異的shRNA都表現出了對過氧化氫引起的細胞凋亡的保護作用。相反，在細胞中過表達TRPM2會導致過氧化氫引起的鈣離子濃度上升的增加和細胞凋亡程度的加重。 這些發現強有力的證明了TRPM2 介導了過氧化氫引起的鈣離子濃度的上升和細胞凋亡。此外，我們還研究了TRPM2激活後的下游事件：半胱氨酸蛋白酶-3，-8和9是否參與到這個過程。我的數據表明過氧化氫誘導細胞凋亡是通過內源和外源通路導致半胱氨酸酶-3激活，而TRPM2在這個過程中起到了重要的決定作用。總括而言，TRPM2 介導了過氧化氫誘導的內皮細胞凋亡，下調內源性的TRPM2的表達會保護血管內皮細胞。 / Reactive Oxygen Species (ROS) play a key role in normal physiological processes such as cell proliferation and growth, as well as in pathological processes. Under pathological conditions ROS contribute to vascular dysfunction and remodeling through oxidative damage. Oxidative stress is now thought to underlie many cardiovascular diseases. Accumulating evidence also demonstrate that many ROS-induced functional abnormalities in the cardiovascular system are preceded by an elevation of intracellular Ca²⁺. / In the first part, I compared the Ca²⁺ responses to ROS between mouse endothelial cells derived from large-sized artey aortas (aortic ECs), and small-sized mesenteric arteries (MAECs). Application of hydrogen peroxide (H₂O₂) caused an increase in cytosolic Ca²⁺ levels ([Ca²⁺]i) in both cell types. The [Ca²⁺]i rises diminished in the presence of U73122, a phospholipase C inhibitor, or Xestospongin C (XeC), an inhibitor for inositol-1,4,5-trisphosphate (IP₃) receptors. In addition, treatment of endothelial cells with H₂O₂ reduced the Ca²⁺ responses to subsequent challenge of ATP. The decreased Ca²⁺ responses to ATP were resulted from a pre-depletion of intracellular Ca²⁺ stores by H₂O₂. Interestingly, we also found that Ca²⁺ store depletion was more sensitive to H₂O₂ treatment in endothelial cells derived from mesenteric arteries than those of derived from aortas. Hypoxanthine-xanthine oxidase (HX-XO) was also found to induce [Ca²⁺]i rises in both types of endothelial cells, the effect of which was mediated by superoxide anions and H₂O₂ but not by hydroxyl radicals. H₂O₂ made a greater contribution to HX-XO-induced [Ca²⁺]i rises in endothelial cells from mesenteric arteries than those from aortas. In summary, H₂O₂ could induce store Ca²⁺ release via phospholipase C-IP₃ pathway in endothelial cells. Emptying of intracellular Ca²⁺ stores contributed to the reduced Ca²⁺ responses to subsequent ATP challenge. Furthermore, the Ca²⁺ responses in endothelial cells of small-sized arteries were more sensitive to H₂O₂ than those of large-sized arteries. / In the second part, I used murine heart microvessel endothelial cell line H5V as a model of endothelial cells from small-sized arteries to investigate the role of Melastatin-like transient receptor potential channel 2 (TRPM2) channels in H₂O₂-induced Ca²⁺ responses and apoptosis. TRPM2 is an oxidant-sensitive cationic non-selective channel that is expressed in mammalian vascular endothelium. A TRPM2 blocking antibody channel (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the Ca²⁺ responses to H₂O₂. Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect in H₂O₂-induced Ca²⁺ responses. H₂O₂-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect on H₂O₂-induced apoptotic cell death. In contrast, overexpression of TRPM2 in H5V cells increased the Ca²⁺ responses to H₂O₂ and aggravated the apoptotic cell death in response to H₂O₂. These findings strongly suggest that the TRPM2 channel mediates Ca²⁺ overload in response to H₂O₂ and contributes to oxidant-induced apoptotic cell death in vascular endothelial cells. I also examined the downstream cascades of TRPM2 activation and explored whether caspase-3, -8 and -9 were involved in this process. My data indicates that H₂O₂-induced cell apoptosis through both intrinsic and extrinsic apoptotic pathways, leading to activation of caspases-3. Furthermore, TRPM2 played an essential role in the process. Together, my data suggest that TRPM2 mediates H₂O₂-induces endothelial cell death and that down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Sun, Lei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 101-114). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Declaration of Originality --- p.I / Abstract --- p.II / 論文摘要 --- p.IV / Acknowledgments --- p.VI / Abbreviations and Units --- p.VII / Table of Contents --- p.IX / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Reactive oxygen species and Reactive nitrogen species --- p.1 / Chapter 1.1.1 --- What is oxidative stress? --- p.1 / Chapter 1.1.2 --- Types of ROS --- p.2 / Chapter 1.1.2.1 --- Hydroxyl radical (*OH) --- p.2 / Chapter 1.1.2.2 --- Hydrogen peroxide (H₂O₂) --- p.3 / Chapter 1.1.2.3 --- Superoxide (O₂*⁻) --- p.4 / Chapter 1.1.2.4 --- Nitric oxide (NO) --- p.5 / Chapter 1.1.3 --- ROS-producing systems --- p.6 / Chapter 1.1.3.1 --- NAD(P)H oxidase --- p.6 / Chapter 1.1.3.2 --- Xanthine oxidase (XO) --- p.7 / Chapter 1.1.3.3 --- The mitochondrial respiratory chain --- p.8 / Chapter 1.1.3.4 --- Uncoupled endothelial NO synthase --- p.8 / Chapter 1.1.4 --- Antioxidant defense mechanisms in the cardiovascular systems --- p.9 / Chapter 1.1.4.1 --- SOD --- p.9 / Chapter 1.1.4.2 --- Catalase --- p.10 / Chapter 1.1.4.3 --- Glutathione peroxidase (GPx) --- p.10 / Chapter 1.1.4.4 --- Small molecules --- p.11 / Chapter 1.1.5 --- Role of oxidative stress in human diseases --- p.12 / Chapter 1.1.6 --- Endothelium dysfunction in oxidative stress-relating human diseases --- p.12 / Chapter 1.1.7 --- Role of Ca²⁺ in oxidative stress-relating human diseases --- p.14 / Chapter 1.1.8 --- Differential effects of ROS on endothelial calcium signaling --- p.15 / Chapter 1.1.8.1 --- Multiple Oxidative Stress-induced Ca²⁺ signaling pathway --- p.16 / Chapter 1.1.9 --- Effects of ROS on Agonist-induced endothelial calcium signaling --- p.19 / Chapter 1.1.10 --- Role of H₂O₂ as EDHF --- p.20 / Chapter 1.1.11 --- Differential effect of ROS on cells derived from large-sized and small-sized artries --- p.21 / Chapter 1.2 --- The transient receptor potential (TRP) Channels --- p.21 / Chapter 1.2.1 --- TRP Channel structure --- p.22 / Chapter 1.2.2 --- TRP Channel function --- p.23 / Chapter 1.2.3 --- TRPM subfamily --- p.23 / Chapter 1.2.3.1 --- TRPM2 Property and Structure --- p.24 / Chapter 1.2.3.2 --- TRPM2 Expression --- p.25 / Chapter 1.2.3.3 --- TRPM2 Activator --- p.25 / Chapter 1.2.3.4 --- TRPM2 Physiological and pathophysiological function --- p.28 / Chapter Chapter 2 --- Objectives of the Present Study --- p.35 / Chapter Chapter 3 --- Materials and methods --- p.37 / Chapter 3.1 --- Ethics statement --- p.37 / Chapter 3.2 --- Materials --- p.37 / Chapter 3.3 --- Methods --- p.38 / Chapter 3.3.1 --- Cell culture --- p.38 / Chapter 3.3.1.1 --- Primary Cell Culture --- p.38 / Chapter 3.3.1.2 --- H5V endothelial cell line --- p.39 / Chapter 3.3.1.3 --- Human embryonic kidney 293 (HEK293) cells --- p.39 / Chapter 3.3.4. --- TRPM2-specific shRNA, TRPM2 and transfection --- p.39 / Chapter 3.3.5 --- Western blotting --- p.40 / Chapter 3.3.6 --- [Ca²⁺]i Studies --- p.43 / Chapter 3.3.6.1 --- Fluo-4/AM- Measuring intracellular [Ca²⁺]i --- p.43 / Chapter 3.3.6.2 --- Fura-2/AM-Measuring intracellular [Ca²⁺]i --- p.44 / Chapter 3.3.6.3 --- Mag-fluo-4-Measuring Ca²⁺ Content in Intracellular Ca²⁺ Stores --- p.45 / Chapter 3.3.7 --- IP₃ measurement --- p.45 / Chapter 3.3.8 --- Electrophysiology --- p.46 / Chapter 3.3.9 --- TRPM2 blocking antibody (TM2E3) and Pre-immune IgG Generation --- p.46 / Chapter 3.3.10 --- DNA fragmentation assay --- p.47 / Chapter 3.3.11 --- DAPI Staining --- p.48 / Chapter 3.3.12 --- MTT assay --- p.48 / Chapter 3.3.13 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Effect of Hydrogen Peroxide and Superoxide Anions on Cytosolic Ca²⁺: Comparison of Endothelial Cells from Large-sized and Small-sized Arteries --- p.50 / Chapter 4.1 --- Introduction --- p.50 / Chapter 4.2 --- Materials and methods --- p.52 / Chapter 4.2.1 --- Primary Cell Culture --- p.52 / Chapter 4.2.2 --- [Ca²⁺]i Measurement --- p.52 / Chapter 4.2.3 --- Measuring Ca²⁺ Content in Intracellular Ca²⁺ Stores --- p.52 / Chapter 4.2.4 --- IP₃ measurement --- p.53 / Chapter 4.2.5 --- Data Analysis --- p.53 / Chapter 4.3 --- Results --- p.53 / Chapter 4.3.1 --- Both Ca²⁺ entry and store Ca²⁺ release contributed to H₂O₂-induced [Ca²⁺]i rises.. --- p.53 / Chapter 4.3.2 --- H₂O₂ enhanced IP₃ production and store Ca²⁺ release --- p.54 / Chapter 4.3.3 --- H₂O₂ reduced the Ca²⁺ responses to ATP in a H₂O₂ concentration and incubation time dependent manner --- p.54 / Chapter 4.3.4 --- H₂O₂ induced Ca²⁺ store depletion --- p.55 / Chapter 4.3.5 --- Ca²⁺ responses to ATP in the absence of H₂O₂ --- p.56 / Chapter 4.3.6 --- Non-involvement of hydroxyl radical --- p.56 / Chapter 4.3.7 --- HX-XO-induced [Ca²⁺]i rises were caused by superoxide anion and hydrogen peroxide --- p.56 / Chapter 4.4 --- Discussion --- p.68 / Chapter Chapter 5 --- Role of TRPM2 in H₂O₂-induced cell apoptosis in endothelial cells --- p.72 / Chapter 5.1 --- Introduction --- p.72 / Chapter 5.2 --- Materials and Methods --- p.73 / Chapter 5.2.1 --- Cell Culture --- p.74 / Chapter 5.2.2 --- [Ca²⁺]i measurement --- p.74 / Chapter 5.2.3 --- DNA fragmentation assay --- p.74 / Chapter 5.2.4 --- MTT assay --- p.74 / Chapter 5.2.5 --- TRPM2-specific shRNA, TRPM2 and transfection --- p.75 / Chapter 5.2.6 --- Electrophysiology --- p.75 / Chapter 5.2.7 --- Western blotting --- p.75 / Chapter 5.2.8 --- DAPI Staining --- p.76 / Chapter 5.2.9 --- Data analysis --- p.76 / Chapter 5.3 --- Results --- p.76 / Chapter 5.3.1 --- Involvement of TRPM2 channels in H₂O₂-induced Ca²⁺ influx in H5V cells --- p.76 / Chapter 5.3.2 --- Involvement of TRPM2 channels in H₂O₂-elicited whole-cell current change in H5V cells --- p.77 / Chapter 5.3.3 --- Role of TRPM2 channels in H₂O₂-induced apoptotic cell death in H5V cells --- p.78 / Chapter 5.3.4 --- Involvement of caspases in H₂O₂-induced apoptotic cell death --- p.79 / Chapter 5.3.5 --- Involvement of TRPM2 in TNF-α-induced cell death in H5V cells --- p.79 / Chapter 5.3 --- Discussion --- p.90 / Chapter Chapter 6 --- General Conclusions, Disscussion and Future work --- p.94 / Chapter 6.1 --- General Conclusions --- p.94 / Chapter 6.2 --- Discussion --- p.95 / Chapter 6.2.1. --- Comparative study --- p.95 / Chapter 6.2.2. --- IP₃ receptor (IP₃R) --- p.95 / Chapter 6.2.3. --- TM2E3-Specific blocking antibody of TRPM2 --- p.95 / Chapter 6.2.4. --- Pathological effect of H₂O₂ at high concentration --- p.96 / Chapter 6.2.5 --- Non-change on Basal [Ca²⁺]i --- p.97 / Chapter 6.3. --- Future work --- p.98 / References --- p.101

血管內膜的進行性增厚是動脈粥樣硬化的重要標誌，並最終導致閉塞性血管病。血管內膜增生的一個主要因素是血管中膜的平滑肌細胞遷移至內膜層並增殖。大量研究證實，在動脈粥樣硬化的發生發展中，過量產生的活性氧簇（ROS）參與了血管壁的增厚。M型瞬時受體電位通道亞家族的成員TRPM2在血管平滑肌細胞中有表達，它能被ROS激活並對Ca²⁺通透，但其在血管平滑肌中的功能以及與心血管疾病的聯繫尚未見報道。 / 本論文著眼於探討TRPM2在鼠和人血管內膜增生中的作用。用血管外周套管法建立在體齧齒類動脈內膜增生模型。套管放置2周後，大鼠股動脈可見明顯的內膜增厚。免疫染色顯示新生內膜及其鄰近中膜區域內有大量增殖細胞核抗原陽性細胞，提示在增生的動脈中，細胞週期活動增強。動脈內膜和中膜内二氫乙錠螢光信號顯著增強，提示了ROS的過量生成。免疫染色和免疫印跡法均顯示，套管損傷導致TRPM2表達上調。免疫螢光雙標TRPM2與α-平滑肌肌動蛋白顯示內膜區域有大量TRPM2陽性的平滑肌細胞。與正常股動脈中膜平滑肌細胞相比，次黃嘌呤和黃嘌呤氧化酶在套管損傷的動脈來源的新生內膜平滑肌細胞中引起更大幅度的細胞內鈣離子濃度升高，而TRPM2抑制性抗體TM2E3預處理可消除這種差異。套管放置3周可引起小鼠頸動脈新生內膜形成，並伴隨著TRPM2表達上調。敲除TRPM2基因可顯著抑制內膜增生。取冠狀動脈搭橋術後殘餘的大隱靜脈，離體培養2周誘導內膜增生。免疫螢光雙標TRPM2與α-平滑肌肌動蛋白顯示新生內膜內含有大量TRPM2陽性的平滑肌細胞。TM2E3和另一TRPM2抑制劑2-氨乙氧基二苯酯硼酸處理均可有效降低內膜的增生。培養齧齒類主動脈平滑肌細胞，用劃痕試驗和MTT法檢測TRPM2阻斷劑和TRPM2基因敲除對過氧化氫誘導的細胞遷移和增殖的影響。結果顯示，暴露於過氧化氫48小時，細胞的遷移和增殖均明顯加快。TM2E3和2-氨乙氧基二苯酯硼酸處理有效抑制過氧化氫誘導的大鼠主動脈平滑肌細胞遷移和增殖；類似地，TRPM2基因敲除可顯著抑制過氧化氫誘導的小鼠主動脈平滑肌細胞遷移和增殖。 / 以上結果表明，血管內膜增生伴隨著TRPM2表達的上調；TRPM2參與了血管內膜增生以及血管平滑肌細胞的遷移、增殖；抑制TRPM2可能是對抗血管內膜增厚的潛在治療手段。 / A hallmark in atherosclerosis is progressive intimal thickening, which leads to occlusive vascular diseases. A causation of neointimal hyperplasia is the migration of medial smooth muscle cells (SMCs) to the intima where they proliferate. It is well recognized that excessive production of reactive oxide species (ROS) contributes to vascular wall thickening during arteriosclerotic development. TRPM2, a member of the melastatin-like transient receptor potential channel subfamily, is a Ca²⁺-permeable cation channel activated by ROS and is expressed in vascular smooth muscle cells (VSMCs). The functional properties of TRPM2 in vascular smooth muscle remain to be identified and an association between TRPM2 and cardiovascular diseases has not been reported. / In the present study, I investigated the involvement of TRPM2 in rodent and human neointimal hyperplasia. In vivo neointimal hyperplasia in rodent arteries was induced by perivascular cuff placement. After the cuff placement for 2 weeks, rat femoral arteries showed distinct intimal thickening. Immunostaining showed a great number of PCNA-positive proliferating cells in the neointima and its adjacent media region, indicating the enhanced cell cycle activity in the hyperplasic arteries. Dihydroethidium signal was markedly increased in the neointima and media of the cuffed arteries, suggesting that ROS is over-produced. Interestingly, both immunostaining and immunoblot showed that cuff-injury also led to an up-regulated expression of TRPM2. Double immunofluorescent labeling of TRPM2 and α-smooth muscle actin showed a large amount of TRPM2-positive SMCs in the neointimal region. Compared with the normal medial SMCs isolated from non-cuffed arteries, the neointimal SMCs from cuff-injured arteries displayed a greater [Ca²⁺] rise in response to hypoxanthine-xanthine oxidase, which was inhibited by pre-treatment with a TRPM2-specific blocking antibody TM2E3. In mouse carotid arteries, cuff placement for 3 weeks caused clear neointimal formation, accompanied by up-regulated expression of TRPM2. Trpm2 disruption dramatically reduced the neointimal growth. Human saphenous vein samples obtained during CABG surgery were organ-cultured for 2 weeks to allow growth of neointima. Double immunofluorescent labeling of TRPM2 and α-smooth muscle actin showed that the neointima contained numerous TRPM2-positive SMCs. Neointimal hyperplasia in the veins was effectively suppressed by in vitro treatment with TM2E3 or a chemical blocker 2-aminoethoxydiphenyl borate. Furthermore, the effect of TRPM2 blockers and Trpm2 disruption on hydrogen peroxide-induced migration and proliferation of cultured rodent aortic SMCs were evaluated by scratch wound healing assay and MTT assay, respectively. It was found that exposure to hydrogen peroxide for 48 hour substantially enhanced the migration and proliferation of rodent aortic SMCs. In rat aortic SMCs, both TM2E3 and 2-aminoethoxydiphenyl borate significantly inhibited the hydrogen peroxide-induced cell migration and proliferation. The hydrogen peroxide-induced cell migration and proliferation of SMCs was also reduced in Trpm2 knockout mice. / Taking together, these results provide strong evidences that in vivo neointimal hyperplasia is accompanied by an up-regulated expression of TRPM2 and that TRPM2 plays a key role in neointimal hyperplasia, VSMCs migration and proliferation. Blocking TRPM2 can be a potential therapeutic approach for protecting blood vessels against intimal thickening. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ru, Xiaochen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 125-151). / Abstracts also in Chinese. / Declaration of Originality --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Acknowledgements --- p.vi / Abbreviations and Units --- p.vii / Table of Content --- p.x / List of Figures --- p.xvi / List of Tables --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Neointimal hyperplasia --- p.1 / Chapter 1.1.1 --- Definition of neointimal hyperplasia --- p.2 / Chapter 1.1.2 --- Medical significance of coronary neointimal hyperplasia --- p.3 / Chapter 1.1.3 --- Pathogenesis of neointimal hyperplasia --- p.5 / Chapter 1.1.3.1 --- “Response to injury“ hypothesis --- p.6 / Chapter 1.1.3.2 --- Role of VSMCs --- p.7 / Chapter 1.1.3.2.1 --- VSMC phenotypic switch --- p.7 / Chapter 1.1.3.2.2 --- Ca²⁺ channel modulation in VSMCs --- p.8 / Chapter 1.1.3.2.3 --- VSMC migration --- p.9 / Chapter 1.1.3.2.4 --- VSMC proliferation --- p.10 / Chapter 1.1.3.2.5 --- Extracellular matrix production by VSMCs --- p.11 / Chapter 1.1.3.3 --- Endothelial dysfunction --- p.11 / Chapter 1.1.3.4 --- Platelet adhesion --- p.12 / Chapter 1.1.3.5 --- Inflammation --- p.13 / Chapter 1.1.4 --- Role of ROS in neointimal hyperplasia --- p.14 / Chapter 1.1.4.1 --- Types of ROS --- p.15 / Chapter 1.1.4.1.1 --- Superoxide anion --- p.16 / Chapter 1.1.4.1.2 --- Hydroxyl radical --- p.16 / Chapter 1.1.4.1.3 --- Hydrogen peroxide --- p.16 / Chapter 1.1.4.1.4 --- Nitric oxide --- p.17 / Chapter 1.1.4.2 --- Sources of ROS in vessel wall --- p.17 / Chapter 1.1.4.3 --- ROS signaling in endothelial cells --- p.19 / Chapter 1.1.4.4 --- ROS signaling in VSMCs --- p.20 / Chapter 1.1.4.5 --- ROS and atherosclerosis --- p.21 / Chapter 1.1.5 --- Current therapeutic approaches to neointimal hyperplasia --- p.23 / Chapter 1.1.5.1 --- Pharmacological approaches --- p.23 / Chapter 1.1.5.2 --- Technical Approaches --- p.25 / Chapter 1.2 --- Transient receptor potential melastatin 2 (TRPM2) channel --- p.27 / Chapter 1.2.1 --- TRP Channels --- p.27 / Chapter 1.2.2 --- TRPM2 structure and expression --- p.29 / Chapter 1.2.2.1 --- Structure --- p.29 / Chapter 1.2.2.2 --- Alternative splicing isoforms --- p.30 / Chapter 1.2.2.3 --- Expression pattern --- p.32 / Chapter 1.2.3 --- TRPM2 channel properties --- p.32 / Chapter 1.2.4 --- TRPM2 activators and inhibitors --- p.32 / Chapter 1.2.4.1 --- Activators --- p.33 / Chapter 1.2.4.1.1 --- ADPR --- p.33 / Chapter 1.2.4.1.2 --- NAD, cADPR and NAADP --- p.33 / Chapter 1.2.4.1.3 --- H₂O₂ and oxidative stress --- p.34 / Chapter 1.2.4.1.4 --- Ca²⁺ --- p.34 / Chapter 1.2.4.1.5 --- Other regulators --- p.35 / Chapter 1.2.4.2 --- Inhibitors --- p.35 / Chapter 1.2.5 --- Biological relevance of TRPM2 --- p.36 / Chapter 1.2.5.1 --- TRPM2 in insulin release --- p.36 / Chapter 1.2.5.2 --- TRPM2 in inflammation --- p.36 / Chapter 1.2.5.3 --- TRPM2 in cell death --- p.37 / Chapter 1.2.5.4 --- TRPM2-mediated lysosomal Ca²⁺ release --- p.38 / Chapter 1.2.5.5 --- TRPM2 and cardiovascular diseases --- p.39 / Chapter Chapter 2 --- Objectives of the Present Study --- p.40 / Chapter Chapter 3 --- Materials and Methods --- p.42 / Chapter 3.1 --- Materials --- p.42 / Chapter 3.1.1 --- Chemicals --- p.42 / Chapter 3.1.2 --- Media, supplements and other reagents for cell/tissue culture --- p.44 / Chapter 3.1.3 --- Antibodies --- p.45 / Chapter 3.1.4 --- Solutions --- p.46 / Chapter 3.1.4.1 --- Solutions for immunohistochemical and immunocytochemical staining --- p.46 / Chapter 3.1.4.2 --- solutions for immunoblotting --- p.47 / Chapter 3.1.4.3 --- Solutions for Genotyping --- p.49 / Chapter 3.1.4.4 --- Solutions for hematoxylin and eosin (HE) staining --- p.50 / Chapter 3.1.4.5 --- Solutions for [Ca²⁺]i measurement --- p.51 / Chapter 3.1.4.6 --- Solutions for IgG purification --- p.51 / Chapter 3.1.5 --- Animals --- p.51 / Chapter 3.1.5.1 --- Rat --- p.51 / Chapter 3.1.5.2 --- Trpm2 knockout mice --- p.52 / Chapter 3.1.5.3 --- Rabbit --- p.52 / Chapter 3.1.5.4 --- Ethics --- p.52 / Chapter 3.1.6 --- Human Tissue --- p.52 / Chapter 3.2 --- Methods --- p.54 / Chapter 3.2.1 --- Rodent models of neointimal hyperplasia --- p.54 / Chapter 3.2.1.1 --- Cuff-induced vascular injury in rat femoral artery --- p.54 / Chapter 3.2.1.2 --- Cuff-induced vascular injury in mouse carotid artery --- p.54 / Chapter 3.2.2 --- Genotyping for Trpm2 knockout mice --- p.55 / Chapter 3.2.2.1 --- Genomic DNA extraction from tail --- p.55 / Chapter 3.2.2.2 --- Polymerase Chain Reaction (PCR) --- p.55 / Chapter 3.2.2.3 --- Agarose gel electrophoresis of DNA --- p.56 / Chapter 3.2.3 --- Human saphenous vein culture and treatment --- p.56 / Chapter 3.2.4 --- Generation of anti-TRPM2 antibody, TRPM2-specific blocking antibody TM2E3 and preimmune IgG --- p.57 / Chapter 3.2.5 --- Histological analysis and immunohistochemistry --- p.58 / Chapter 3.2.6 --- Western blotting --- p.59 / Chapter 3.2.7 --- Detection of ROS production by dihydroethidium fluorescence --- p.60 / Chapter 3.2.8 --- Isolation of rodent neointimal and medial smooth muscle cells --- p.60 / Chapter 3.2.9 --- Culture of rodent aortic smooth muscle cells --- p.61 / Chapter 3.2.9.1 --- Cell culture --- p.61 / Chapter 3.2.9.2 --- Cell identification --- p.61 / Chapter 3.2.10 --- [Ca²⁺]i measurement --- p.62 / Chapter 3.2.11 --- Cell proliferation assay --- p.63 / Chapter 3.2.12 --- Cell migration assay --- p.63 / Chapter 3.2.13 --- Statistical analysis --- p.64 / Chapter Chapter 4 --- ROS over-production and TRPM2 up-regulation in cuff-induced rodent neointimal hyperplasia --- p.65 / Chapter 4.1 --- Introduction --- p.65 / Chapter 4.2 --- Materials and Methods --- p.66 / Chapter 4.2.1 --- Cuff-induced vascular injury in rat femoral artery --- p.66 / Chapter 4.2.2 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.66 / Chapter 4.2.3 --- Histological analysis and immunohistochemistry --- p.66 / Chapter 4.2.4 --- Western blotting --- p.67 / Chapter 4.2.5 --- Detection of ROS production --- p.67 / Chapter 4.2.6 --- Isolation of rat neointimal and medial smooth muscle cells --- p.68 / Chapter 4.2.7 --- [Ca²⁺]i measurement --- p.68 / Chapter 4.2.8 --- Statistical analysis --- p.68 / Chapter 4.3 --- Results --- p.69 / Chapter 4.3.1 --- Cuff-induced neointimal hyperplasia in rat femoral arteries --- p.69 / Chapter 4.3.2 --- ROS over-production in neointimal region of cuff-injured rat femoral arteries --- p.69 / Chapter 4.3.3 --- TRPM2 up-regulation in neointimal region of cuff-injured rat femoral arteries --- p.69 / Chapter 4.3.4 --- Enhanced [Ca²⁺]i response to HX-XO in rat neointimal smooth muscle cells --- p.70 / Chapter 4.4 --- Discussion --- p.81 / Chapter Chapter 5 --- TRPM2 contributes to human and rodent neointimal hyperplasia --- p.86 / Chapter 5.1 --- Introduction --- p.86 / Chapter 5.2 --- Materials and Methods --- p.87 / Chapter 5.2.1 --- Cuff-induced vascular injury in mouse carotid artery --- p.87 / Chapter 5.2.2 --- Genotyping for Trpm2 knockout mice --- p.87 / Chapter 5.2.3 --- Organ culture of human saphenous vein --- p.87 / Chapter 5.2.4 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.88 / Chapter 5.2.5 --- Histological analysis and immunohistochemistry --- p.88 / Chapter 5.2.6 --- Western blotting --- p.88 / Chapter 5.2.7 --- Isolation of mouse neointimal and medial smooth muscle cells --- p.89 / Chapter 5.2.8 --- [Ca²⁺]i measurement --- p.89 / Chapter 5.2.9 --- Statistical analysis --- p.90 / Chapter 5.3 --- Results --- p.90 / Chapter 5.3.1 --- Cuff-induced neointimal hyperplasia was reduced in Trpm2 knockout mice --- p.90 / Chapter 5.3.2 --- [Ca²⁺]i response to HX-XO in mouse neointimal smooth muscle cells --- p.90 / Chapter 5.3.3 --- Inhibiting TRPM2 reduced the neointimal hyperplasia in in vitro cultured human saphenous vein --- p.91 / Chapter 5.4 --- Discussion --- p.99 / Chapter Chapter 6 --- Role of TRPM2 in H₂O₂-stimulated migration and proliferation of vascular smooth muscle cells --- p.103 / Chapter 6.1 --- Introduction --- p.103 / Chapter 6.2 --- Materials and Methods --- p.104 / Chapter 6.2.1 --- Culture of rodent aortic smooth muscle cells --- p.104 / Chapter 6.2.2 --- Immunocytochemistry --- p.104 / Chapter 6.2.3 --- Genotyping for Trpm2 knockout mice --- p.104 / Chapter 6.2.4 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.104 / Chapter 6.2.5 --- [Ca²⁺]i measurement --- p.105 / Chapter 6.2.6 --- Cell proliferation assay --- p.105 / Chapter 6.2.7 --- Western blotting --- p.105 / Chapter 6.2.8 --- Cell migration assay --- p.106 / Chapter 6.2.9 --- Statistical analysis --- p.106 / Chapter 6.3 --- Results --- p.106 / Chapter 6.3.1 --- H₂O₂-induced [Ca²⁺]i rises in rodent aortic smooth muscle cells --- p.106 / Chapter 6.3.2 --- Role of TRPM2 in H₂O₂-stimulated smooth muscle cell proliferation --- p.107 / Chapter 6.3.3 --- Role of TRPM2 in H₂O₂-stimulated smooth muscle cell migration --- p.108 / Chapter 6.4 --- Discussion --- p.118 / Chapter Chapter 7 --- General Conclusion and Future Work --- p.121 / Chapter 7.1 --- Concluding remarks --- p.121 / Chapter 7.2 --- Future work --- p.123 / Chapter 7.2.1 --- Specific downstream signaling pathway of TRPM2 that mediates ROS-induced VSMC proliferation and migration --- p.123 / Chapter 7.2.2 --- Involvement of TRPM2 in leukocyte infiltration and inflammation in vascular wall --- p.124 / References --- p.125 / List of Publications --- p.152

Many endothelial cell (EC) functions depend on influx of extracellular Ca2+, which is triggered by a variety of mechanical and chemical signals. Here, we discuss possible pathways for this Ca2+ entry. The superfamily of cation channels derived from the "transient receptor potential" (TRP) channels is introduced. Several members of this family are expressed in ECs, and they provide pathways for Ca2+ entry. All TRP subfamilies may contribute to the Ca2+ entry channels or to the regulation of Ca2+ entry in EC. Members of Ca2+ entry channels in endothelium probably belong to the canonical TRP subfamily, TRPC. All TRPC1-6 have been discussed as Ca2+ entry channels that might be store-operated and/or receptor-operated. More importantly, knockout models of TRPC4 have proven that this channel is functionally involved in the regulation of endothelial-dependent vasorelaxation and in the control of EC barrier function. TRPC1 might be an important candidate for involvement of eodothelial growth factors. TRPC3 is unequivocally important for a sustained EC Ca2+ entry. ECs express different patterns of TRPCs, which may increase the variability of TRPC channel function by formation of different multiheteromers. Among the two other TRP subfamilies, TRPMV and TRPM, at least TRPV4 and TRPM4 are EC channels. TRPV4 is a Ca2+ entry channel that is activated by an increase in cell volume, which might be involved in mechano-sensing, by an increase in temperature, and perhaps by ligand-activation. TRPM4 is a nonselective cation channel, which is not Ca2+ permeable. It is probably modulated by NO and might be essential for regulating the inward driving force for Ca2+ entry. Possible modes of TRP channel regulation are described, involving (a) activation via the phospholipase (PL)Cβ and PLCγ pathways; (b) activation by lipids (diacylglycerol [DAG], arachidonic acid); (c) Ca2+ depletion of Ca2+ stores in the endoplasmic reticulum; (d) shear stress; and (e) radicals.

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

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