麦冬正丁醇提取部位调控人脐静脉血管内皮细胞凋亡相关基因与信号转导通路研究

目的:研究麦冬正丁醇提取部位对H_2O_2造模的人脐静脉血管内皮细胞(HUVEC)凋亡相关基因和信号转导通路调控,探讨其对HUVEC保护作用的分子机制。方法:用H_2O_2构建HUVEC凋亡模型,运用MTT检测细胞增殖情况,流式细胞仪检测凋亡相关基因Bc1-2表达,免疫组化方法观察NF-kB表达情况。结果:麦冬正丁醇提取部位可以减少H_2O_2造模的HUVEC的凋亡,促进Bc1-2的表达,并能降低NF-kB的表达。结论:麦冬正丁醇提取部位可以通过NF-kB路径,促进凋亡抑制基因Bc1-2的表达,从而起到抗HUVEC凋亡的作用。     

麦冬不同提取物对过氧化氢损伤人血管内皮细胞ICAM-1、VEGF、Bcl-2表达的影响

目的:观察麦冬不同提取物对过氧化氢诱导的人脐静脉内皮细胞(HUVEC)间黏附分子-1(ICAM-1)和VEGF、Bc(?)-2表达的影响。方法:体外培养HUVEC,用过氧化氢(H_2O_2)制造HUVEC损伤模型。以四甲基偶氮唑蓝(MTT)比色法检测细胞存活数量,用流式细胞仪检测HUVEC表面ICAM-1的表达量;免疫细胞化学方法检测HUVEC的VEGF、Bc(?)-2的分布情况。结果:模型组较正常对照组细胞增殖活性明显降低(P<0.01)。与模型组相比,经麦冬水提物、正丁醇提取物处理组细胞增殖活性明显增加(P<0.05,P<0.01)。流式细胞仪检测显示正丁醇提取物可降低过氧化氢增加的ICAM-1基因的表达。Bc(?)-2的表达,模型组明显低于正常对照组,而正丁醇组表达明显高于模型组(P<0.01)。VEGF的表达,模型组明显高于正常对照组,麦冬水提物、正丁醇提取物处理组高于模型组(P<0.05,P<0.01)。结论:麦冬提取物具有抗凋亡、促增殖、降低细胞间黏附分子-1表达的作用,尤以正丁醇提取物效果更为显著。     

黄芪皂苷Ⅳ对H_2O_2诱导的心肌细胞凋亡的保护作用

目的:探讨黄芪皂苷对H2O2诱导的心肌细胞凋亡的保护作用。方法:以H2O2诱导SD大鼠心肌损伤细胞模型为基础,用黄芪皂苷Ⅳ预处理进行干预。MTT法检测不同时段细胞凋亡情况,Western blot和RT-PCR检测24h时段Cyclin D1蛋白和mRNA表达水平。结果:H2O2对SD大鼠心肌细胞的损伤呈时间依赖性。H2O2可显著诱导SD大鼠心肌细胞凋亡,而这一作用可被黄芪皂苷Ⅳ显著抑制。结论:黄芪皂苷Ⅳ对H2O2诱导的SD大鼠心肌细胞损伤有明显保护作用。     

麦冬不同提取物对过氧化氢损伤人血管内皮细胞ICAM-1、VEGF、Bcl-2 表达的影响

目的:观察麦冬不同提取物对过氧化氢诱导的人脐静脉内皮细胞(HUVEC)间黏附分子-1(ICAM-1)和VEGF、Bcl-2表达的影响。方法:体外培养HUVEC,用过氧化氢(H202)制造HUVEC损伤模型。以四甲基偶氮唑蓝(MTT)比色法检测细胞存活数量,用流式细胞仪检测HUVEC表面ICAM-1的表达量;免疫细胞化学方法检测HUVEC的VEGF、Bcl-2的分布情况。结果:模型组较正常对照组细胞增殖活性明显降低(P<0.01)。与模型组相比,经麦冬水提物、正丁醇提取物处理组细胞增殖活性明显增加(P<0.05,P<0.01)。流式细胞仪检测显示正丁醇提取物可降低过氧化氢增加的ICAM-1基因的表达。Bcl-2的表达,模型组明显低于正常对照组,而正丁醇组表达明显高于模型组(P<0.01)。VEGF的表达,模型组明显高于正常对照组,麦冬水提物、正丁醇提取物处理组高于模型组(P<0.05,P<0.01)。结论:麦冬提取物具有抗凋亡、促增殖、降低细胞间黏附分子-1表达的作用,尤以正丁醇提取物效果更为显著。     

黄芪皂苷IV对H2O2诱导的心肌细胞凋亡的保护作用

目的:探讨黄芪皂苷对H2O2诱导的心肌细胞凋亡的保护作用.方法:以H2O2诱导SD大鼠心肌损伤细胞模型为基础,用黄芪皂苷IV预处理进行干预.MTT法检测不同时段细胞凋亡情况,Western blot和RT-PCR检测24h时段Cyclin D1蛋白和mRNA表达水平.结果:H2O2对SD大鼠心肌细胞的损伤呈时间依赖性.H2O2可显著诱导SD大鼠心肌细胞凋亡.而这一作用可被黄芪皂苷IV显著抑制.结论:黄芪皂苷IV对H2O2诱导的SD大鼠心肌细胞损伤有明显保护作用.     

Sestrin2对热暴露肺上皮细胞凋亡的干预作用及其机制*

目的:探讨Sestrin2蛋白对热暴露肺上皮细胞凋亡的干预作用及其作用机制。方法:体外培养的Beas-2B细胞分为对照组(37℃)和热暴露组(39℃、40℃和41℃),在上述温度中暴露不同时间(0、3、6和12 h),胰酶消化后收集细胞,分别通过Western blot、荧光分光光度计、流式细胞仪等方法检测细胞中的Sestrin2、超氧化物歧化酶(SOD)、活性氧自由基(ROS)表达水平,细胞线粒体膜电位及细胞凋亡率。基因序列克隆入高表达质粒pcDNA 3.1⁺中,采用Lipfectamine 2000方法转染Beas-2B细胞,构建Sestrin2和SOD高表达细胞,观察细胞线粒体膜电位及细胞凋亡等指标的变化。结果:随着暴露温度的升高,与对照组相比,热暴露组细胞Sestrin2蛋白表达水平下降。在41℃热暴露Beas-2B细胞,不同时间点ROS水平显著上升,线粒体膜电位显著下降,细胞凋亡率增加。Sestrin2和SOD高表达细胞,在41℃暴露条件下,与对照组比较,ROS表达水平显著降低,线粒体膜电位下降幅度减小,热暴露导致细胞凋亡率降低。结论: Sestrin2能够通过线粒体膜电位和SOD缓解热暴露引起肺上皮细胞的凋亡,对Beas-2B细胞具有保护作用。     

黄芪皂苷Ⅳ对H2O2诱导的心肌细胞凋亡的保护作用

目的：探讨黄芪皂苷对H2O2诱导的心肌细胞凋亡的保护作用。方法：以H2O2诱导SD大鼠心肌损伤细胞模型为基础,用黄芪皂苷Ⅳ预处理进行干预。MTT法检测不同时段细胞凋亡情况,Western blot和RT-PCR检测24h时段Cyclin D1蛋白和mRNA表达水平。结果：H2O2对SD大鼠心肌细胞的损伤呈时间依赖性。H2O2可显著诱导SD大鼠心肌细胞凋亡,而这一作用可被黄芪皂苷Ⅳ显著抑制。结论：黄芪皂苷Ⅳ对H2O2诱导的SD大鼠心肌细胞损伤有明显保护作用。     

高糖诱导血管内皮细胞凋亡机制的研究

目的:探讨高糖诱导血管内皮细胞凋亡的分子机制。方法:在高浓度葡萄糖的培养基中培养人脐静脉内皮细胞(human umbilical vein endothelium cells,HUVECs),模拟高糖血症条件下内皮细胞的病理状态。通过MTT检测HUVEC细胞生存情况;JC-1检测HUVEC细胞线粒体膜电位;逆转录-聚合酶链反应和荧光素酶报告基因检测己糖激酶Ⅱ(hexokinaseⅡ,HKⅡ)的转录;免疫印迹和免疫共沉淀检测VDAC1、HKⅡ、Bcl-2、Bax线粒体上蛋白表达水平和它们之间的相互作用。结果:25 m M和100m M葡萄糖诱导HUVEC细胞生存率分别下降了19.21%±4.13%和25.29%±5.78%;线粒体膜电位分别降低了34.19%±5.13%和58.63%±4.78%;HKⅡ蛋白表达水平分别降低了13.97%±6.32%和35.13%±5.18%;使得HKⅡ同VDAC1互作减弱,代偿性增强了VDAC1同Bax互作。结论:高糖下调HUVEC细胞HKⅡ表达水平,增强线粒体膜通透性,最终诱导了细胞凋亡。     

麦冬不同提取物对过氧化氢损伤人血管内皮细胞ICAM-1、VEGF、Bcl-2表达的影响

目的：观察麦冬不同提取物对过氧化氢诱导的人脐静脉内皮细胞（HUVEC）间黏附分子-1（ICAM-1）和VEGF、Bcl-2表达的影响。方法：体外培养HUVEC,用过氧化氢（H2O2）制造HUVEC损伤模型。以四甲基偶氮唑蓝（MTT）比色法检测细胞存活数量,用流式细胞仪检测HUVEC表面ICAM-1的表达量;免疫细胞化学方法检测HUVEC的VEGF、Bcl-2的分布情况。结果：模型组较正常对照组细胞增殖活性明显降低（P〈0．01）。与模型组相比,经麦冬水提物、正丁醇提取物处理组细胞增殖活性明显增加（P〈0．05,P〈0．01）。流式细胞仪检测显示正丁醇提取物可降低过氧化氢增加的ICAM-1基因的表达。Bcl-2的表达,模型组明显低于正常对照组,而正丁醇组表达明显高于模型组（P〈0．01）。VEGF的表达,模型组明显高于正常对照组,麦冬水提物、正丁醇提取物处理组高于模型纽（P〈0．05,P〈0．01）。结论：麦冬提取物具有抗凋亡、促增殖、降低细胞间黏附分子-1表达的作用,尤以正丁醇提取物效果更为显著。     

去氢木香内酯干预人乳腺癌MCF-7细胞机制的探究

目的:探讨去氢木香内酯对乳腺癌MCF-7细胞凋亡、线粒体跨膜电位及代谢物的影响,为研究去氢木香内酯诱导MCF-7细胞凋亡的作用机制提供新的视角。方法:采用流式细胞仪测定不同浓度去氢木香内酯(0、2、4、8μg/m L)对MCF-7细胞凋亡及线粒体跨膜电位的影响;GC-TOFMS测定去氢木香内酯作用前后,MCF-7细胞内具有显著性变化的代谢差异物。结果:研究结果表明,去氢木香内酯能诱导MCF-7细胞的凋亡、促进线粒体跨膜电位的降低;正交偏最小二乘法判别分析(OPLS-DA)多维统计方法对代谢组学数据分析得到柠檬酸、D-核糖、脯氨酸、苯丙氨酸、赖氨酸等16种代谢差异物。结论:推测去氢木香内酯通过引起线粒体跨膜电位降低而破坏了线粒体的结构,进一步阻碍了线粒体的功能,导致了细胞内代谢物的紊乱,最终诱导了细胞的凋亡。     

Glutathione oxidation in calcium- and p38 MAPK-dependent membrane blebbing of endothelial cells

Under conditions where apoptosis is prevented, peroxides disrupt the endothelial monolayer by inducing cytoskeletal rearrangements, cell retraction and formation of arrays of membrane blebs. In human umbilical vein endothelial cells (HUVEC), the H(2)O(2)-induced membrane blebbing was found to be a transient process executed by two parallel signaling mechanisms: (i) mobilization of cytosolic [Ca(2+)](i) through a pathway requiring oxidation of reduced glutathione (GSH), and (ii) activation of p38 mitogen-activated protein kinases (MAPK) independently of GSH oxidation and Ca(2+) mobilization. In the HUVEC, membrane blebbing was thus blocked by inhibition of GSH oxidation, Ca(2+) mobilization or p38 MAPK activation. Stimulation of GSH peroxidation with ebselen potentiated the H(2)O(2)-induced oscillating Ca(2+) response and the bleb formation, but not p38 phosphorylation. Chelation of [Ca(2+)](i) abolished the blebbing process but not p38 activation. In addition, in the GSH peroxidase-resistant cell line ECV304, H(2)O(2) was unable to promote membrane blebbing or significant Ca(2+) release, while p38 became phosphorylated. However, [Ca(2+)](i) was increased and blebs were formed, when the ECV304 were treated with ebselen before H(2)O(2). Together, this leads to a model where oxidative stress, through both Ca(2+)-dependent and p38 kinase-mediated phosphorylation events, causes reassembly of the actin cytoskeleton and subsequent appearance of membrane blebs at the plasma membrane.     

Anti-apoptotic and anti-senescence effects of Klotho on vascular endothelial cells

Klotho-mutated mice manifest multiple age-related disorders that are observed in humans. A recent study suggested that Klotho protein might function as an anti-aging hormone in mammals. Because it has been reported that apoptosis and senescence in vascular endothelial cells are closely related to the progression of atherosclerosis, we investigated Klotho's ability to interfere with apoptosis and cellular senescence in human umbilical vascular endothelial cells (HUVEC). Klotho overexpression decreased H(2)O(2)-induced apoptosis in COS-1 cells and Jurkat cells. Klotho protein also reduced H(2)O(2)- and etoposide-induced apoptosis in HUVEC. Caspase-3 and caspase-9 activity was lower in Klotho-treated HUVEC than in control cells. Senescence-associated beta-gal staining showed that Klotho protein interferes with H(2)O(2)-induced premature cellular senescence. The expression of p53 and p21 was lower in Klotho-treated cells. Our study suggests that Klotho acts as a humoral factor to reduce H(2)O(2)-induced apoptosis and cellular senescence in vascular cells.     

Mono-(2-Ethylhexyl) Phthalate Induces Injury in Human Umbilical Vein Endothelial Cells

Mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate (DEHP), is a widespread environmental contaminant and has been proved to have potential adverse effects on the reproductive system, carcinogenicity, liver, kidney and developmental toxicities. However, the effect of MEHP on vascular system remains unclear. The main purpose of this study was to evaluate the cytotoxic effects of MEHP on human umbilical endothelial cells (HUVEC) and its possible molecular mechanism. HUVEC cells were treated with MEHP (0, 6.25, 12.5, 25,50 and 100 µM), and the cellular apoptosis and mitochondrial membrane potential as well as intracellular reactive oxygen species were determined. In present study, MEHP induced a dose-dependent cell injury in HUVEC cell via an apoptosis pathway as characterized by increased percentage of sub-G1, activation of caspase-3, -8and -9, and increased ratio of Bax/bcl-2 mRNA and protein expression as well as cytochrome C releasing. In addition, there was obvious oxidative stress, represented by decreased glutathione level, increased malondialdehyde level and superoxide dismutase activity. N-Acetylcysteine, as an antioxidant that is a direct reactive oxygen species scavenger, could effectively block MEHP-induced reactive oxygen species generation, mitochondrial membrane potential loss and cell apoptosis. These data indicated that MEHP induced apoptosis in HUVEC cells through a reactive oxygen species-mediated mitochondria-dependent pathway.     

H2O2-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells

This study employed confocal laser scanning microscopy to monitor the effect of H2O2 on cytosolic as well as mitochondrial calcium (Ca2+) concentrations, mitochondrial inner membrane potential (psi m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H2O2, in the absence of extracellular Ca2+ ([Ca2+],) led to an increase either in cytosolic and in mitochondrial Ca2+ concentration. Additionally, H2O2 induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H2O2 on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H2O2. However, the H2O2-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca2+ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H2O2-evoked changes in mitochondrial Ca2+ concentration but not those in membrane potential and FAD state. The present results have indicated that H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas.     

Hydrogen peroxide removes TRPM4 current desensitization conferring increased vulnerability to necrotic cell death

Necrosis is associated with an increase in plasma membrane permeability, cell swelling, and loss of membrane integrity with subsequent release of cytoplasmic constituents. Severe redox imbalance by overproduction of reactive oxygen species is one of the main causes of necrosis. Here we demonstrate that H(2)O(2) induces a sustained activity of TRPM4, a Ca(2+)-activated, Ca(2+)-impermeant nonselective cation channel resulting in an increased vulnerability to cell death. In HEK 293 cells overexpressing TRPM4, H(2)O(2) was found to eliminate in a dose-dependent manner TRPM4 desensitization. Site-directed mutagenesis experiments revealed that the Cys(1093) residue is crucial for the H(2)O(2)-mediated loss of desensitization. In HeLa cells, which endogenously express TRPM4, H(2)O(2) elicited necrosis as well as apoptosis. H(2)O(2)-mediated necrosis but not apoptosis was abolished by replacement of external Na(+) ions with sucrose or the non-permeant cation N-methyl-d-glucamine and by knocking down TRPM4 with a shRNA directed against TRPM4. Conversely, transient overexpression of TRPM4 in HeLa cells in which TRPM4 was previously silenced re-established vulnerability to H(2)O(2)-induced necrotic cell death. In addition, HeLa cells exposed to H(2)O(2) displayed an irreversible loss of membrane potential, which was prevented by TRPM4 knockdown.     

Calcium-Mediated Mitochondrial Permeability Transition Involved in Hydrogen Peroxide-Induced Apoptosis in Tobacco Protoplasts

In the present study, we focused on whether Intracellular free Ca^2＋ （[Ca^2＋],） regulates the formation of mltochondrlal permeability transition pore （MPTP） In H2O2-induced apoptosis In tobacco protoplasts. It was shown that the decrease In mltochondrlal membrane potential （△ψm） preceded the appearance of H2O2-Induced apoptosls; pretreatment with the specific MPTP Inhibitor cyclosporine A, which also Inhibits Ca^2＋ cycling by the mitochondria, effectively retarded apoptosls and the decrease In △ψm. Apoptosls and decreased △ψm were exacerbated by CaCl2, whereas the plasma membrane voltage-dependent Ca^2＋ channel blocker lanthanum chloride （LaCl3） attentuated these responses. Chelation of extracellular Ca^2＋ with EGTA almost totally Inhibited apoptosls and the decrease In △ψmInduced by H2O2. The time-course of changes In [Ca^2＋]l In apoptosls was detected using the Ca^2＋ probe Fiuo-3 AM. These studies showed that [Ca^2＋]1 was Increased at the very early stage of H2O2-Induced apoptosls. The EGTA evidently Inhibited the Increase In [Ca^2＋]1 Induced by H=O=, whereas It was only partially Inhibited by LaCl3. The results suggest that H2O2 may elevate cytoplasmic free Ca^2＋ concentrations In tobacco protoplasts, which mainly results from the entry of extracellular Ca^2＋, to regulate mltochondrlal permeability transition. The signaling pathway of [Ca^2＋]1-medlated mltochondrlal permeability transition was associated with H2O2-Induced apoptosis In tobacco protoplaete.     

A novel butyrolactone derivative inhibited smooth muscle cell migration and proliferation and maintained endothelial cell functions through selectively affecting Na, K-ATPase activity and mitochondria membrane potential during in vitro angiogenesis

We have found that 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran -2(3H)-one (3BDO), could effectively suppress human umbilical vascular endothelial cell (HUVEC) apoptosis induced by deprivation of fibroblast growth factor-2 and serum. Here, our purpose was to investigate whether 3BDO could modulate angiogenesis and its possible acting mechanism. The effect of 3BDO on angiogenesis was investigated by capillary-like tubule formation and rat aortic ring assay. Proliferation and migration of cells were detected by counting living cell number and scraping cell monolayer, respectively. Na, K-ATPase activity was measured spectrophotometrically. Mitochondrial membrane potential was analyzed using tetramethylrhodamine methylester fluorescence by confocal microscopy. Our results showed that 3BDO inhibited migration and proliferation of vascular smooth muscle cells (VSMCs), but maintained migration and tubule formation of HUVECs. In HUVECs, 3BDO inhibited Na, K-ATPase activity, but had no effect on mitochondria membrane potential. In VSMCs, it did not affect Na, K-ATPase activity, but depressed mitochondria membrane potential obviously. The data showed that 3BDO had selective effects on HUVECs and VSMCs, it might perform its role through the selective effects on the activity of Na, K-ATPase and the mitochondria membrane potential in HUVECs and VSMCs.     

Effect of H2O2 on CCK-8-evoked changes in mitochondrial activity in isolated mouse pancreatic acinar cells

BACKGROUND INFORMATION: This paper studies the effect of H(2)O(2) on mitochondrial responses evoked by CCK-8 (cholecystokinin 8) in mouse pancreatic acinar cells. Cytosolic ([Ca(2+)](c)) and mitochondrial ([Ca(2+)](m)) free-calcium concentrations, mitochondrial inner membrane potential (psi(m)) and FAD autofluorescence were monitored using confocal laser scanning microscopy. RESULTS: CCK-8 induced an increase in [Ca(2+)](m) that slowly declined towards the prestimulation level. Depolarization of psi(m) that partially recovered, as well as increases in FAD autofluorescence, could also be observed in response to the hormone. Pretreatment of cells with 1 mM H(2)O(2) alone resulted in marked changes in mitochondrial parameters and, moreover, H(2)O(2) inhibited the CCK-8-evoked changes in [Ca(2+)](m), psi(m) and FAD autofluorescence. The results of the present study have demonstrated that CCK-8 can evoke marked changes in pancreatic acinar cell mitochondrial activity and that CCK-8-evoked responses are blocked by H(2)O(2). Additionally, H(2)O(2) releases Ca(2+) from intracellular stores and inhibits pancreatic acinar cell responses to CCK-8. CONCLUSION: The effects observed reflect an impairment of mitochondrial activity in the presence of H(2)O(2) that could represent some of its mechanisms of action to induce cellular damage leading to cell dysfunction and generation of pathologies.     

Hydrogen peroxide induces apoptosis in human hepatoma cells and alters cell redox status

Direct exposure of human hepatoma cell line SMMC-7721 to hydrogen peroxide (H2O2) can induce apoptosis. Apoptosis induced by H2O2 was inhibited by cycloheximide, actinomycin D, 3-aminobenzamide, EGTA or Zn2+. H2O2 can increase the level of intracellular Ca2+, downregulate GSH levels, slightly induce lipid peroxidation, and lead to change in the ratio of reduced ion components to oxidized ion components of cells. Analysis of flow cytometry indicates that H2O2 decreases the level of Bcl-2. The data indicate that H2O2-induced apoptosis requires new mRNA and protein syntheses; H2O2 can activate Ca2+/Mg2+-dependent endonuclease leading to internucleosomal DNA fragmentation and activation of poly (ADP-ribose) polymerase interfering with the energy metabolism of the cell. The H2O2 downregulation of GSH may be more important for apoptosis than H2O2 induction of lipid peroxidation, and the H2O2 induced changes in redox status of the cell may be among the original events which lead up to other biochemical changes.     

Role of TRPM2 in H(2)O(2)-Induced Cell Apoptosis in Endothelial Cells

Melastatin-like transient receptor potential channel 2 (TRPM2) is an oxidant-sensitive and cationic non-selective channel that is expressed in mammalian vascular endothelium. Here we investigated the functional role of TRPM2 channels in hydrogen peroxide (H(2)O(2))-induced cytosolic Ca(2+) ([Ca(2+)](i)) elavation, whole-cell current increase, and apoptotic cell death in murine heart microvessel endothelial cell line H5V. A TRPM2 blocking antibody (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the [Ca(2+)](i) rise and whole-cell current change in response to H(2)O(2). Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect. H(2)O(2)-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 against H(2)O(2)-induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF)-α-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca(2+)](i) and whole-cell currents to H(2)O(2). TRPM2 overexpression also aggravated the H(2)O(2)-induced apoptotic cell death. Downstream pathways following TRPM2 activation was examined. Results showed that TRPM2 activity stimulated caspase-8, caspase-9 and caspase-3. These findings strongly suggest that TRPM2 channel mediates cellular Ca(2+) overload in response to H(2)O(2) and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells. Down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.