Role of transient receptor potential melastatin 2 in neointimal hyperplasia, vascular smooth muscle cell migration and proliferation

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.iAbstract --- p.ii論文摘要 --- p.ivAcknowledgements --- p.viAbbreviations and Units --- p.viiTable of Content --- p.xList of Figures --- p.xviList of Tables --- p.xviiiChapter Chapter 1 --- Introduction --- p.1Chapter 1.1 --- Neointimal hyperplasia --- p.1Chapter 1.1.1 --- Definition of neointimal hyperplasia --- p.2Chapter 1.1.2 --- Medical significance of coronary neointimal hyperplasia --- p.3Chapter 1.1.3 --- Pathogenesis of neointimal hyperplasia --- p.5Chapter 1.1.3.1 --- “Response to injury“ hypothesis --- p.6Chapter 1.1.3.2 --- Role of VSMCs --- p.7Chapter 1.1.3.2.1 --- VSMC phenotypic switch --- p.7Chapter 1.1.3.2.2 --- Ca²⁺ channel modulation in VSMCs --- p.8Chapter 1.1.3.2.3 --- VSMC migration --- p.9Chapter 1.1.3.2.4 --- VSMC proliferation --- p.10Chapter 1.1.3.2.5 --- Extracellular matrix production by VSMCs --- p.11Chapter 1.1.3.3 --- Endothelial dysfunction --- p.11Chapter 1.1.3.4 --- Platelet adhesion --- p.12Chapter 1.1.3.5 --- Inflammation --- p.13Chapter 1.1.4 --- Role of ROS in neointimal hyperplasia --- p.14Chapter 1.1.4.1 --- Types of ROS --- p.15Chapter 1.1.4.1.1 --- Superoxide anion --- p.16Chapter 1.1.4.1.2 --- Hydroxyl radical --- p.16Chapter 1.1.4.1.3 --- Hydrogen peroxide --- p.16Chapter 1.1.4.1.4 --- Nitric oxide --- p.17Chapter 1.1.4.2 --- Sources of ROS in vessel wall --- p.17Chapter 1.1.4.3 --- ROS signaling in endothelial cells --- p.19Chapter 1.1.4.4 --- ROS signaling in VSMCs --- p.20Chapter 1.1.4.5 --- ROS and atherosclerosis --- p.21Chapter 1.1.5 --- Current therapeutic approaches to neointimal hyperplasia --- p.23Chapter 1.1.5.1 --- Pharmacological approaches --- p.23Chapter 1.1.5.2 --- Technical Approaches --- p.25Chapter 1.2 --- Transient receptor potential melastatin 2 (TRPM2) channel --- p.27Chapter 1.2.1 --- TRP Channels --- p.27Chapter 1.2.2 --- TRPM2 structure and expression --- p.29Chapter 1.2.2.1 --- Structure --- p.29Chapter 1.2.2.2 --- Alternative splicing isoforms --- p.30Chapter 1.2.2.3 --- Expression pattern --- p.32Chapter 1.2.3 --- TRPM2 channel properties --- p.32Chapter 1.2.4 --- TRPM2 activators and inhibitors --- p.32Chapter 1.2.4.1 --- Activators --- p.33Chapter 1.2.4.1.1 --- ADPR --- p.33Chapter 1.2.4.1.2 --- NAD, cADPR and NAADP --- p.33Chapter 1.2.4.1.3 --- H₂O₂ and oxidative stress --- p.34Chapter 1.2.4.1.4 --- Ca²⁺ --- p.34Chapter 1.2.4.1.5 --- Other regulators --- p.35Chapter 1.2.4.2 --- Inhibitors --- p.35Chapter 1.2.5 --- Biological relevance of TRPM2 --- p.36Chapter 1.2.5.1 --- TRPM2 in insulin release --- p.36Chapter 1.2.5.2 --- TRPM2 in inflammation --- p.36Chapter 1.2.5.3 --- TRPM2 in cell death --- p.37Chapter 1.2.5.4 --- TRPM2-mediated lysosomal Ca²⁺ release --- p.38Chapter 1.2.5.5 --- TRPM2 and cardiovascular diseases --- p.39Chapter Chapter 2 --- Objectives of the Present Study --- p.40Chapter Chapter 3 --- Materials and Methods --- p.42Chapter 3.1 --- Materials --- p.42Chapter 3.1.1 --- Chemicals --- p.42Chapter 3.1.2 --- Media, supplements and other reagents for cell/tissue culture --- p.44Chapter 3.1.3 --- Antibodies --- p.45Chapter 3.1.4 --- Solutions --- p.46Chapter 3.1.4.1 --- Solutions for immunohistochemical and immunocytochemical staining --- p.46Chapter 3.1.4.2 --- solutions for immunoblotting --- p.47Chapter 3.1.4.3 --- Solutions for Genotyping --- p.49Chapter 3.1.4.4 --- Solutions for hematoxylin and eosin (HE) staining --- p.50Chapter 3.1.4.5 --- Solutions for [Ca²⁺]i measurement --- p.51Chapter 3.1.4.6 --- Solutions for IgG purification --- p.51Chapter 3.1.5 --- Animals --- p.51Chapter 3.1.5.1 --- Rat --- p.51Chapter 3.1.5.2 --- Trpm2 knockout mice --- p.52Chapter 3.1.5.3 --- Rabbit --- p.52Chapter 3.1.5.4 --- Ethics --- p.52Chapter 3.1.6 --- Human Tissue --- p.52Chapter 3.2 --- Methods --- p.54Chapter 3.2.1 --- Rodent models of neointimal hyperplasia --- p.54Chapter 3.2.1.1 --- Cuff-induced vascular injury in rat femoral artery --- p.54Chapter 3.2.1.2 --- Cuff-induced vascular injury in mouse carotid artery --- p.54Chapter 3.2.2 --- Genotyping for Trpm2 knockout mice --- p.55Chapter 3.2.2.1 --- Genomic DNA extraction from tail --- p.55Chapter 3.2.2.2 --- Polymerase Chain Reaction (PCR) --- p.55Chapter 3.2.2.3 --- Agarose gel electrophoresis of DNA --- p.56Chapter 3.2.3 --- Human saphenous vein culture and treatment --- p.56Chapter 3.2.4 --- Generation of anti-TRPM2 antibody, TRPM2-specific blocking antibody TM2E3 and preimmune IgG --- p.57Chapter 3.2.5 --- Histological analysis and immunohistochemistry --- p.58Chapter 3.2.6 --- Western blotting --- p.59Chapter 3.2.7 --- Detection of ROS production by dihydroethidium fluorescence --- p.60Chapter 3.2.8 --- Isolation of rodent neointimal and medial smooth muscle cells --- p.60Chapter 3.2.9 --- Culture of rodent aortic smooth muscle cells --- p.61Chapter 3.2.9.1 --- Cell culture --- p.61Chapter 3.2.9.2 --- Cell identification --- p.61Chapter 3.2.10 --- [Ca²⁺]i measurement --- p.62Chapter 3.2.11 --- Cell proliferation assay --- p.63Chapter 3.2.12 --- Cell migration assay --- p.63Chapter 3.2.13 --- Statistical analysis --- p.64Chapter Chapter 4 --- ROS over-production and TRPM2 up-regulation in cuff-induced rodent neointimal hyperplasia --- p.65Chapter 4.1 --- Introduction --- p.65Chapter 4.2 --- Materials and Methods --- p.66Chapter 4.2.1 --- Cuff-induced vascular injury in rat femoral artery --- p.66Chapter 4.2.2 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.66Chapter 4.2.3 --- Histological analysis and immunohistochemistry --- p.66Chapter 4.2.4 --- Western blotting --- p.67Chapter 4.2.5 --- Detection of ROS production --- p.67Chapter 4.2.6 --- Isolation of rat neointimal and medial smooth muscle cells --- p.68Chapter 4.2.7 --- [Ca²⁺]i measurement --- p.68Chapter 4.2.8 --- Statistical analysis --- p.68Chapter 4.3 --- Results --- p.69Chapter 4.3.1 --- Cuff-induced neointimal hyperplasia in rat femoral arteries --- p.69Chapter 4.3.2 --- ROS over-production in neointimal region of cuff-injured rat femoral arteries --- p.69Chapter 4.3.3 --- TRPM2 up-regulation in neointimal region of cuff-injured rat femoral arteries --- p.69Chapter 4.3.4 --- Enhanced [Ca²⁺]i response to HX-XO in rat neointimal smooth muscle cells --- p.70Chapter 4.4 --- Discussion --- p.81Chapter Chapter 5 --- TRPM2 contributes to human and rodent neointimal hyperplasia --- p.86Chapter 5.1 --- Introduction --- p.86Chapter 5.2 --- Materials and Methods --- p.87Chapter 5.2.1 --- Cuff-induced vascular injury in mouse carotid artery --- p.87Chapter 5.2.2 --- Genotyping for Trpm2 knockout mice --- p.87Chapter 5.2.3 --- Organ culture of human saphenous vein --- p.87Chapter 5.2.4 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.88Chapter 5.2.5 --- Histological analysis and immunohistochemistry --- p.88Chapter 5.2.6 --- Western blotting --- p.88Chapter 5.2.7 --- Isolation of mouse neointimal and medial smooth muscle cells --- p.89Chapter 5.2.8 --- [Ca²⁺]i measurement --- p.89Chapter 5.2.9 --- Statistical analysis --- p.90Chapter 5.3 --- Results --- p.90Chapter 5.3.1 --- Cuff-induced neointimal hyperplasia was reduced in Trpm2 knockout mice --- p.90Chapter 5.3.2 --- [Ca²⁺]i response to HX-XO in mouse neointimal smooth muscle cells --- p.90Chapter 5.3.3 --- Inhibiting TRPM2 reduced the neointimal hyperplasia in in vitro cultured human saphenous vein --- p.91Chapter 5.4 --- Discussion --- p.99Chapter Chapter 6 --- Role of TRPM2 in H₂O₂-stimulated migration and proliferation of vascular smooth muscle cells --- p.103Chapter 6.1 --- Introduction --- p.103Chapter 6.2 --- Materials and Methods --- p.104Chapter 6.2.1 --- Culture of rodent aortic smooth muscle cells --- p.104Chapter 6.2.2 --- Immunocytochemistry --- p.104Chapter 6.2.3 --- Genotyping for Trpm2 knockout mice --- p.104Chapter 6.2.4 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.104Chapter 6.2.5 --- [Ca²⁺]i measurement --- p.105Chapter 6.2.6 --- Cell proliferation assay --- p.105Chapter 6.2.7 --- Western blotting --- p.105Chapter 6.2.8 --- Cell migration assay --- p.106Chapter 6.2.9 --- Statistical analysis --- p.106Chapter 6.3 --- Results --- p.106Chapter 6.3.1 --- H₂O₂-induced [Ca²⁺]i rises in rodent aortic smooth muscle cells --- p.106Chapter 6.3.2 --- Role of TRPM2 in H₂O₂-stimulated smooth muscle cell proliferation --- p.107Chapter 6.3.3 --- Role of TRPM2 in H₂O₂-stimulated smooth muscle cell migration --- p.108Chapter 6.4 --- Discussion --- p.118Chapter Chapter 7 --- General Conclusion and Future Work --- p.121Chapter 7.1 --- Concluding remarks --- p.121Chapter 7.2 --- Future work --- p.123Chapter 7.2.1 --- Specific downstream signaling pathway of TRPM2 that mediates ROS-induced VSMC proliferation and migration --- p.123Chapter 7.2.2 --- Involvement of TRPM2 in leukocyte infiltration and inflammation in vascular wall --- p.124References --- p.125List of Publications --- p.15