Cytotoxic Power of Hydrogen Peroxide Effect on Endothelial Cells in vitro

The currently existing terms “apoptotic index” and “apoptotic potential” are insufficient for the adequate qualitative evaluation and analysis of the proapoptotic activity of chemical agents within the wide range of therapeutic and toxic doses. Herein we introduce a new concept of cytotoxic power as an alternative to these terms related to apoptosis and other types of cell death on the basis of experimental data of hydrogen peroxide cytotoxic activity towards the human umbilical vein endothelial cells (HUVEC). We also propose an algorithm for computing cytotoxic power. Correlations between the percentage of early apoptotic and late apoptotic/necrotic cells and the expression or concentration of endothelial markers such as CD143, CD309, von Willebrand factor, phosphatase PTEN, have been revealed.     

过氧化氢对内皮祖细胞凋亡相关蛋白表达的影响

目的:比较不同浓度过氧化氢(hydrogen peroxide,H2O2)对人外周血来源的内皮祖细胞(endothelial progenitor cells,EPCs)生存能力的改变及其对凋亡相关蛋白表达的影响。方法:采用密度梯度离心法和差速贴壁法从人外周静脉血中分离培养内皮祖细胞。选取传代后第3代EPCs作为研究对象,以终浓度分别为50μmol/L、100μmol/L、150μmol/L、200μmol/L、300μmol/L和400μmol/L的过氧化氢处理内皮祖细胞12 h,同时设立正常处理对照组。CCK-8法检测各组内皮祖细胞生存能力的差异;Western blot分析各组内皮祖细胞中凋亡相关蛋白Bax、Bcl-2和p53的蛋白表达情况。结果:与对照组相比,在浓度为50μmol/L、100μmol/L和150μmol/L过氧化氢处理组中细胞存活能力逐渐增强,促凋亡蛋白Bax、p53随之下调,抗凋亡蛋白Bcl-2则显著上调;在浓度为200μmol/L、300μmol/L和400μmol/L过氧化氢处理组中细胞生存能力相对于正常对照组逐渐减弱,促凋亡蛋白Bax、p53表达水平逐渐增加,抗凋亡蛋白Bcl-2表达水平下降。结论:过氧化氢对内皮祖细胞存活能力和凋亡相关蛋白Bax、Bcl-2、p53表达的影响均呈双相性变化。     

Specific Growth Rate Plays a Critical Role in Hydrogen Peroxide Resistance of the Marine Oligotrophic Ultramicrobacterium Sphingomonas alaskensis Strain RB2256

The marine oligotrophic ultramicrobacterium Sphingomonas alaskensis RB2256 has a physiology that is distinctly different from that of typical copiotrophic marine bacteria, such as Vibrio angustum S14. This includes a high level of inherent stress resistance and the absence of starvation-induced stress resistance to hydrogen peroxide. In addition to periods of starvation in the ocean, slow, nutrient-limited growth is likely to be encountered by oligotrophic bacteria for substantial periods of time. In this study we examined the effects of growth rate on the resistance of S. alaskensis RB2256 to hydrogen peroxide under carbon or nitrogen limitation conditions in nutrient-limited chemostats. Glucose-limited cultures of S. alaskensis RB2256 at a specific growth rate of 0.02 to 0.13 h⁻¹ exhibited 10,000-fold-greater viability following 60 min of exposure to 25 mM hydrogen peroxide than cells growing at a rate of 0.14 h⁻¹ or higher. Growth rate control of stress resistance was found to be specific to carbon and energy limitation in this organism. In contrast, V. angustum S14 did not exhibit growth rate-dependent stress resistance. The dramatic switch in stress resistance that was observed under carbon and energy limitation conditions has not been described previously in bacteria and thus may be a characteristic of the oligotrophic ultramicrobacterium. Catalase activity varied marginally and did not correlate with the growth rate, indicating that hydrogen peroxide breakdown was not the primary mechanism of resistance. More than 1,000 spots were resolved on silver-stained protein gels for cultures growing at rates of 0.026, 0.076, and 0.18 h⁻¹. Twelve protein spots had intensities that varied by more than twofold between growth rates and hence are likely to be important for growth rate-dependent stress resistance. These studies demonstrated the crucial role that nutrient limitation plays in the physiology of S. alaskensis RB2256, especially under oxidative stress conditions.     

Role of Hydroxyl Radicals in Escherzchza Colz Killing Induced by Hydrogen Peroxide

Escherichia coli lethality by hydrogen peroxide is characterized by two modes of killing. In this paper we have found that hydroxyl radicals (OH -) generated by H₂O₂ and intracellular divalent iron are not involved in the induction of mode one lethality (i.e. cell killing produced by concentrations of H₂O₂ lower than 2.5 mM). In fact, the OH radical scavengers, thiourea, ethanol and dimethyl sulfoxide, and the iron chelator, desferrioxarnine, did not affect the survival of cells exposed to 2.5mM H₂O₂. In addition cell vulnerability to the same H₂O₂ concentration was independent on the intracellular iron content. In contrast, mode two lethality (i.e. cell killing generated by concentrations of H₂O₂ higher than 10mM) was markedly reduced by OH radical scavengers and desferrioxamine and was augmented by increasing the intracellular iron content.

It is concluded that OH. are required for mode two killing of E. coli by hydrogen peroxide.     

H2O2致体外培养动脉内皮细胞损伤及山莨菪碱保护

目的:实验以过氧化氢(H2O2)损伤体外培养动脉内皮细胞(AEC),对过氧化氢介导内皮细胞损伤、凋亡的机制进行探讨,观察山莨菪碱(Ani)是否抑制H2O2介导的AEC损伤、凋亡,并探讨Ani保护损伤、凋亡的机制.方法:体外培养AEC随机分组:对照组(正常培养AEC);H2O2损伤组(0.5 mmol/L H2O2损伤12 h);Ani组(不同浓度Ani:0.05,0.1,0.2 mg/mL处理45 min后加0.5 mmol/L H2O2损伤12 h).结果:1.低浓度H2O2(0.5 mmol/L)可以损伤AEC并诱导凋亡.H2O2损伤组台盼蓝着染率及LDH释放率均高于对照组(P<0.01);细胞总抗氧化能力(T-AOC)下降(比对照组P<0.01);琼脂糖凝胶电泳发现凋亡细胞的梯状电泳条带;细胞荧光染色见凋亡形态特征.2.Ani对H2O2诱导的内皮细胞损伤具保护作用.Ani组与H2O2损伤组比较,台盼蓝着染率及LDH释放率下降;T-AOC上升;在Ani 0.2 mg/mL组DNA琼脂糖凝胶电泳未见凋亡细胞特征性的梯状电泳条带;荧光染色未见凋亡细胞特征.结论:1.低浓度过氧化氢使AEC总抗氧化能力明显下降,耗竭细胞抗氧化能力,可致AEC的损伤、凋亡.2.山莨菪碱能减少AEC抗氧化能力的消耗,维持抗氧化能力平衡,保护0.5 mmol/L H2O2 介导的AEC损伤、凋亡.     

Cytoglobin Up-regulated by Hydrogen Peroxide Plays a Protective Role in Oxidative Stress

Cytoglobin (Cygb) is a recently discovered intracellular respiratory globin, which exists in all types of cells. It has been suggested that Cygb has a role in protecting cells against oxidative stress. In the present study we have tested this hypothesis. The N2a neuroblastoma cells were exposed to various kinds of insults, including hydrogen peroxide (H₂O₂), hypoxia, kainic acid, high extracellular CaCl₂, high osmolarity, UV irradiation and heat shock. Among them, only H₂O₂-treatment induced a significant up-regulation of cytoglobin mRNA level. We stably transfected N2a cells with Cygb-siRNA vectors and successfully knocked down Cygb. The Cygb-siRNA could exacerbate cell death upon H₂O₂-treatment, as demonstrated by MTT cell viability assay. Thus, Cygb in neuronal cells might be specifically induced under oxidative stress to protect them from death.     

拟南芥AtDAD1 超量表达植株对H2O2抗性的研究

构建拟南芥AtDAD1超量表达载体,以农杆菌介导的方法转化拟南芥哥伦比亚生态型,比较AtDAD1超量表达植株和野生型植株表现型的差异,以及两者对H2O2抗性的不同。实验显示,AtDAD1转基因拟南芥生长较野生型拟南芥更为强壮,对高浓度H2O2有较强的耐受力。测定两者糖含量,发现AtDAD1转基因拟南芥叶片糖的含量明显高于野生型拟南芥叶片。以上结果表明,AtDAD1基因可能参与植物生长发育,并可能在拟南芥抵抗凋亡的过程中发挥重要的作用。     

Hydrogen Peroxide Metabolism in Yeasts

A catalase-negative mutant of the yeast Hansenula polymorpha consumed methanol in the presence of glucose when the organism was grown in carbon-limited chemostat cultures. The organism was apparently able to decompose the H₂O₂ generated in the oxidation of methanol by alcohol oxidase. Not only H₂O₂ generated intracellularly but also H₂O₂ added extracellularly was effectively destroyed by the catalase-negative mutant. From the rate of H₂O₂ consumption during growth in chemostat cultures on mixtures of glucose and H₂O₂, it appeared that the mutant was capable of decomposing H₂O₂ at a rate as high as 8 mmol · g of cells⁻¹ · h⁻¹. Glutathione peroxidase (EC 1.11.1.9) was absent under all growth conditions. However, cytochrome c peroxidase (CCP; EC 1.11.1.5) increased to very high levels in cells which decomposed H₂O₂. When wild-type H. polymorpha was grown on mixtures of glucose and methanol, the CCP level was independent of the rate of methanol utilization, whereas the level of catalase increased with increasing amounts of methanol in the substrate feed. Also, the wild type decomposed H₂O₂ at a high rate when cells were grown on mixtures of glucose and H₂O₂. In this case, an increase of both CCP and catalase was observed. When Saccharomyces cerevisiae was grown on mixtures of glucose and H₂O₂, the level of catalase remained low, but CCP increased with increasing rates of H₂O₂ utilization. From these observations and an analysis of cell yields under the various conditions, two conclusions can be drawn. (i) CCP is a key enzyme of H₂O₂ detoxification in yeasts. (ii) Catalase can effectively compete with mitochondrial CCP for hydrogen peroxide only if hydrogen peroxide is generated at the site where catalase is located, namely in the peroxisomes.     

Hydrogen Peroxide in Human Blood

Blood and plasma of humans and rats were analyzed for hydrogen peroxide. The samples were analyzed after deproteinization with trichloroacetic acid, immediately after they were withdrawn from human volunteers or rats. A radio-isotopic technique based on peroxide-dependent decarboxylation of 1-¹⁴C-alpha-ketoacids and consequent liberation of ¹⁴CO₂ was used. The results demonstrate the presence ofmicromolar levels of H₂O₂, both, in the plasma as well as in the whole blood. The values in the whole blood were substantially greater than the plasma. This was true for rats as well as humans. The presence of such significant quantities of H₂O₂ in the blood have been demonstrated for the first time. The investigation, therefore, opens a newer avenue of research on diseases purported to be related to the generation of oxygen radicals in vivo.     

Role of Nitric Oxide and Hydrogen Peroxide During the Salt Resistance Response

Ion homeostasis is essential for plant cell resistance to salt stress. Under salt stress, to avoid cellular damage and nutrient deficiency, plant cells need to maintain adequate K nutrition and a favorable K to Na ratio in the cytosol. Recent observations revealed that both nitric oxide (NO) and hydrogen peroxide (H₂O₂) act as signaling molecules to regulate K to Na ratio in calluses from Populus euphratica under salt stress. Evidence indicated that NO mediating H₂O₂ causes salt resistance via the action of plasma membrane H⁺-ATPase but that activity of plasma membrane NADPH oxidase is dependent on NO. Our study demonstrated the signaling transduction pathway. In this addendum, we proposed a testable hypothesis for NO function in regulation of H₂O₂ mediating salt resistance.Key Words: hydrogen peroxide, nitric oxide, signaling molecule, salt resistanceUnder salinity conditions, tolerant plant cells achieve ion homeostasis by extruding Na to the external medium and/or compartmentalizing into vacuoles, maintaining K uptake and high K and low Na in the cytosol.^1,2 Control of Na movement across the plasma membrane (PM) and tonoplast in order to maintain a low Na concentration in the cytoplasm is a key factor of cellular adaptation to salt stress.^3,4 Na transport across the PM is dependent on the electrochemical gradient created by the PM H⁺-ATPase.^5,6 It has been proven that the activity of the PM H⁺-ATPase is a key index of plant adaptation to salt stress.⁷ Therefore, the regulation of expression of the PM H⁺-ATPase may represent an important cellular mechanism for salt resistance. In contrast to our understanding of the regulation of PM H⁺-ATPase by other factors, the roles of NO and H₂O₂ act as signals under salt stress have been less known.Previous studies have shown that both NO and H₂O₂ function as stress signals in plants, mediating a range of resistance mechanisms in plants under stress conditions.^8–10 We have previously shown that NO serves as a signal in inducing salt resistance by increasing the K to Na ratio, which is dependent on the increased PM H⁺-ATPase activity in calluses from reed.¹¹ Although NO acts as a signal molecule under salt stress and induces salt resistance by increasing PM H⁺-ATPase activity, our research results also indicated NO can not activate purified PM H⁺-ATPase activity, at least in vitro. Subsequently, we set out to find the other signal molecules and factors between NO and PM H⁺-ATPase activity. Since our studies have indicated that NO can not induce salt resistance directly, what roles dose it play in salt resistance in tolerant cells under salt stress? We initially hypothesized ABA or H₂O₂ might be downstream signal molecules to regulate the activity of PM H⁺-ATPase. Further results indicated H₂O₂ content increased greatly under salt stress. Since H₂O₂ might be the candidate downstream signal molecule, we tested PM H⁺-ATPase activity and K to Na ratio in calluses by adding H₂O₂. The results suggested that H₂O₂ inducing an increased PM H⁺-ATPase activity resulted in an increased K to Na ratio. Summing up this new assay that allows us to speculate NO maybe regulate the H₂O₂ generation.Since H₂O₂ is involved in downstream signal molecule of NO, PM NADPH oxidase, the main source of H₂O₂ production, might be the regulated target of NO. We took a pharmacological approach to examine the speculation. The results indicated that PM NADPH oxidase is required for H₂O₂ accumulation and PM NADPH oxidase activity could attribute to NO in calluses under salt stress. These results also raised another question regarding what concentrations of NO can induce such effects. In our experiments, NO content was induced 1.6 times higher than the control values under salt treatment. We speculated there exists an effective balance point in NO signal system similar to previous reports by Delledonne et al.¹² in disease resistance.Further research work is required to decipher the mechanism through which NO and H₂O₂ acts and how K and Na elements uptake might be connected with salt resistance. We would like to propose a simple testable model that accounts for the results reported in this paper (Fig. 1). According to our model, H₂O₂ rather than NO is the major signaling molecular that mediated directly PM H⁺-ATPase under salt stress. Normally, NO generated from nitric oxide synthase (NOS) acts as a signal molecule to regulate other mechanisms. Under salt stress, accumulated NO activates PM NADPH oxidase activity. Then, a number of H₂O₂ is produced from PM NADPH oxidase. The PM H⁺-ATPase is activated greatly by the accumulated H₂O₂. Eventually, the transmembrane electrochemical gradient is created and K to Na ratio increases. The model we have proposed here is testable and should provide further insights into salt resistance mechanism regulated by NO and H₂O₂ signal molecules.Open in a separate windowFigure 1Hypothetical model for the potential function of NO and H₂O₂ as signaling molecules in inducing salt resistance. Salt stress activates a signal transduction cascade that leads to the increased activity of PM H⁺-ATPase, whose expression produces salt resistance. NO is generated by NOS, and H₂O₂ is produced by NADPH oxidase attributed to NO. The activity of PM H⁺-ATPase is regulated by H₂O₂ directly under salt stress. The model is based on the recent results in calluses from P. euphratica¹² and those previously reported on the NO function in reed.¹¹Research on roles of NO and H₂O₂ under stress conditions in plant is advancing rapidly. Further analysis of salt resistance mechanism with novel technology will certainly increase our knowledge in this field.     

内皮前体细胞的动员与调节

胚胎发生时期,内皮前体细胞(endothelial progenitor cells,EPCs)参与了原始血管形成的最初过程(血管发生)。已有的证据显示,分化为内皮细胞(endothelial cells,Ecs)的前体也存在于成人中,正常情况下,EPCs停留在成人的骨髓,但是,可以通过细胞因子或血管生成因子信号被动员到循环血,迁移到生理或病理条件下的新血管形成位点,并原位分化成内皮细胞,快速和及时地修复损伤的血管。自源的EPCs原住动员或移植是治疗性血管再生的一个潜在、有效的方法,因此,探究EPCs从骨髓的动员和调节,对血管再生以及修复器官功能具有重要的意义。     

过氧化氢在水杨酸诱导的蚕豆气孔关闭中的作用

许多植物病原菌可通过气孔进入叶片组织,因此减小气孔开度有利于提高植物的抗性。我们通过表皮条分析和激光扫描共聚显微镜得到的证据表明在保卫细胞中过氧化氢可能是水杨酸信号的中间环节。SA可以浓度依赖的方式诱导气孔关闭（图1A）,H2O2也有类似的作用（图1B）。100μmol/L的水杨酸诱导的气孔关闭作用可明显地被20U/ml的过氧化氢酶或10μmol/L的Vc逆转,但CAT和Vc单独处理时诱导气孔开放的作用很微弱。单细胞中基于荧光探针DCFH的时间进程实验表明直接外加（图版I）或显微注射100μmol/L的SA均可诱导保卫细胞中H2O2产生,但以显微注射双蒸水作为对照时对DCFH荧光无影响（图版II）。这些结果暗示了植物被病原菌感染时可能通过产生H2O2导致气孔关闭而阻止病原菌继续通过气孔侵入。     

Hydrogen Peroxide Modification of Human Oxyhemoglobin

The effect of H₂O₂ on the primary structure of OxyHb was studied. Upon treatment of Oxy Hb with H₂O₂ ([Heme]/[H₂O₂] =I), tryptophan and methionine residues of the /-chain were modified. Treatment of ApoHb with H₂O₂ resulted in the modification of histidine and methionine residues in both globin chains. Tryptophan residues were unaffected. Modification of methionine residues in both the β-chain of OxyHb and ApoHb probably results from the direct oxidation of mcthionine by H₂O₂. The modification of histidine residues in ApoHb may be mediated by a metal-catalyzed oxidation system comprised of H₂O₂ and histidine-bound iron. The H₂O₂-mediated modification of tryptophan in the OxyHb β-chain. however, requires the heme moiety.     

Hydrogen Peroxide Modification of Human Oxyhemoglobin

The effect of H₂O₂ on the primary structure of OxyHb was studied. Upon treatment of Oxy Hb with H₂O₂ ([Heme]/[H₂O₂] =I), tryptophan and methionine residues of the /-chain were modified. Treatment of ApoHb with H₂O₂ resulted in the modification of histidine and methionine residues in both globin chains. Tryptophan residues were unaffected. Modification of methionine residues in both the β-chain of OxyHb and ApoHb probably results from the direct oxidation of mcthionine by H₂O₂. The modification of histidine residues in ApoHb may be mediated by a metal-catalyzed oxidation system comprised of H₂O₂ and histidine-bound iron. The H₂O₂-mediated modification of tryptophan in the OxyHb β-chain. however, requires the heme moiety.     

Critical Role of Tight Junctions in Drug Delivery across Epithelial and Endothelial Cell Layers

Epithelia in multicellular organisms constitute the frontier that separates the individual from the environment. Epithelia are sites of exchange as well as barriers, for the transit of ions and molecules from and into the organism. Therapeutic agents, in order to reach their target, frequently need to cross epithelial and endothelial sheets. Two routes are available for such purpose: the transcellular and the paracellular pathways. The former is employed by lipophilic drugs and by molecules selectively transported by channels, pumps and carriers present in the plasma membrane. Hydrophilic molecules cannot cross biological membranes, therefore their transepithelial transport could be significantly enhanced if they moved through the paracellular pathway. Transit through this route is regulated by tight junctions (TJs). The discovery in recent years of the molecular mechanisms of the TJ has allowed the design of different procedures to open the paracellular route in a reversible manner. These strategies could be used to enhance drug delivery across epithelial and endothelial barriers. The procedures employed include the use of peptides homologous to external loops of integral TJ proteins, silencing the expression of TJ proteins with antisense oligonucleotides and siRNAs as well as the use of toxins and proteins derived from microorganisms that target TJ proteins.