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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328254 |
| Date | January 2012 |
| Contributors | Sun, Lei, Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xiv, 114 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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