Role of reactive oxygen species and gp91phox in endothelial dysfunction of pulmonary arteries induced by chronic hypoxia

1. This study investigates the role of nitric oxide (NO) and reactive oxygen species (ROS) on endothelial function of pulmonary arteries in a mice model of hypoxia-induced pulmonary hypertension. 2. In pulmonary arteries from control mice, the NO-synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) potentiated contraction to prostaglandin F2alpha (PGF2alpha) and completely abolished relaxation to acetylcholine. In extrapulmonary but not intrapulmonary arteries, acetylcholine-induced relaxation was slightly inhibited by polyethyleneglycol-superoxide dismutase (PEG-SOD) or catalase. 3. In pulmonary arteries from hypoxic mice, ROS levels (evaluated using dihydroethidium staining) were higher than in controls. In these arteries, relaxation to acetylcholine (but not to sodium nitroprusside) was markedly diminished. L-NAME abolished relaxation to acetylcholine, but failed to potentiate PGF2-induced contraction. PEG-SOD or catalase blunted residual relaxation to acetylcholine in extrapulmonary arteries, but did not modify it in intrapulmonary arteries. Hydrogen peroxide elicited comparable (L-NAME-insensitive) relaxations in extra- and intrapulmonary arteries from hypoxic mice. 4. Exposure of gp91phox(-/-) mice to chronic hypoxia also decreased the relaxant effect of acetylcholine in extrapulmonary arteries. However, in intrapulmonary arteries from hypoxic gp91phox(-/-) mice, the effect of acetylcholine was similar to that obtained in mice not exposed to hypoxia. 5. Chronic hypoxia increases ROS levels and impairs endothelial NO-dependent relaxation in mice pulmonary arteries. Mechanisms underlying hypoxia-induced endothelial dysfunction differ along pulmonary arterial bed. In extrapulmonary arteries from hypoxic mice, endothelium-dependent relaxation appears to be mediated by ROS, in a gp91phox-independent manner. In intrapulmonary arteries, endothelial dysfunction depends on gp91phox, the latter being rather the trigger than the mediator of impaired endothelial NO-dependent relaxation     

Ostreolysin induces sustained contraction of porcine coronary arteries and endothelial dysfunction in middle- and large-sized vessels

Ostreolysin (Oly), a cytolytic and cardiotoxic protein from the oyster mushroom (Pleurotus ostreatus), is lethal for mice with an LD₅₀ of 1170 μg/kg following intravenous application. Its cardiotoxicity is associated with hyperkalemia, which is probably a consequence of potassium released from the lysed cells. Moreover, sub-micromolar concentrations of Oly induce a concentration-dependent increase in rat aortic ring tension, suggesting that ischaemia, and consequent hypoxic injury of cardiomyocytes, could also derive from vasospasm induced by this toxic protein.The purpose of the present study was to demonstrate histopathological lesions caused by Oly after parenteral application to rats, and to define the mechanisms of Oly-induced vasoconstriction using inhibitors verapamil, lanthanum chloride, and selective endothelin receptor antagonist TBC3214, which have different molecular targets, in vitro on porcine coronary artery rings. We found that Oly causes endothelial injury with perivascular oedema in the heart and lungs, as well as myocardial haemorrhages in rats. Treatment of porcine coronary artery rings with Oly causes concentration-dependent vasoconstriction and prevents endothelium-mediated relaxation. Using TBC3214 as a selective blocker of the endothelin A receptor, we showed that vasoconstriction induced by Oly was independent of endothelin release and its effects. Verapamil (1 μM) greatly reduced Oly-evoked contractions of porcine coronary artery rings, while lanthanum abolished them completely. These results provide evidence that the contraction of coronary arteries by Oly is due mainly to the increased influx of Ca²⁺ from the extracellular space through voltage-dependent L-type Ca²⁺ channels and cation non-selective channels. Experiments suggest that Oly damages endothelial cells both in vitro and in vivo, and probably exhibits direct contractile effects on coronary smooth muscle cells.     

The role of reactive oxygen species in the modulation of the contraction induced by angiotensin II in carotid artery from diabetic rat

The modulation played by reactive oxygen species on the angiotensin II-induced contraction in type I-diabetic rat carotid was investigated. Concentration-response curves for angiotensin II were obtained in endothelium-intact or endothelium-denuded carotid from control or streptozotocin-induced diabetic rats, pre-treated with tiron (superoxide scavenger), PEG-catalase (hydrogen peroxide scavenger), dimethylthiourea (hydroxyl scavenger), apocynin [NAD(P)H oxidase inhibitor], SC560 (cyclooxygenase-1 inhibitor), SC236 (cyclooxygenase-2 inhibitor) or Y-27632 (Rho-kinase inhibitor). Reactive oxygen species were measured by flow cytometry in dihydroethidium (DHE)-loaded endothelial cells. Cyclooxygenase and AT(1)-receptor expression was assessed by immunohistochemistry. Diabetes increased the angiotensin II-induced contraction but reduced the agonist potency in rat carotid. Endothelium removal, tiron or apocynin restored the angiotensin II-induced contraction in diabetic rat carotid to control levels. PEG-catalase, DMTU or SC560 reduced the angiotensin II-induced contraction in diabetic rat carotid at the same extent. SC236 restored the angiotensin II potency in diabetic rat carotid. Y-27632 reduced the angiotensin II-induced contraction in endothelium-intact or -denuded diabetic rat carotid. Diabetes increased the DHE-fluorescence of carotid endothelial cells. Apocynin reduced the DHE-fluorescence of endothelial cells from diabetic rat carotid to control levels. Diabetes increased the muscular cyclooxygenase-2 expression but reduced the muscular AT(1)-receptor expression in rat carotid. In summary, hydroxyl radical, hydrogen peroxide and superoxide anion-derived from endothelial NAD(P)H oxidase mediate the hyperreactivity to angiotensin II in type I-diabetic rat carotid, involving the participation of cyclooxygenase-1 and Rho-kinase. Moreover, increased muscular cyclooxygenase-2 expression in type I-diabetic rat carotid seems to be related to the local reduced AT(1)-receptor expression and the reduced angiotensin II potency.     

Voltage- and time-dependent inhibitory effects on rat aortic and porcine coronary artery contraction induced by propafenone and quinidine.

1. Class I antiarrhythmic drugs (e.g. Na+ channel blockers) such as propafenone and quinidine also inhibit voltage-gated Ca2+ and K+ channels. In the present paper the voltage- and time-dependent inhibitory effects of propafenone and quinidine were studied on depolarization-induced vascular contractions and 45Ca2+ uptake in isolated endothelium denuded rat aorta and pig left descending coronary artery. 2. Quinidine and propafenone (10(-7) M -5 x 10(-5) M) produced a concentration-dependent relaxation of the contractions induced by 80 mM KCl. Propafenone was significantly more potent (P < 0.05) than quinidine in both rat aorta and pig coronary arteries but both drugs more potent (P < 0.05) in relaxing rat aorta than pig coronary arteries. In rat aortic rings, the relaxant effects of propafenone were unaffected by pretreatment with the Na+ channel blocker, tetrodotoxin. 3. The degree of inhibition produced after prolonged exposure (40 min) to propafenone and quinidine differed as the time of depolarization with 80 mM KCl was increased. Quinidine (3 x 10(-6) M, 10(-5) M and 3 x 10(-5) M) not only produced an inhibition at the very early stage of contraction, but also a time-dependent inhibition was observed. In contrast, propafenone (10(-6) M, 3 x 10(-6) M and 10(-5) M) produced a more marked concentration-dependent early block but only a mild time-dependent inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell permeability, migration, and reactive oxygen species induced by multiwalled carbon nanotubes in human microvascular endothelial cells

Multiwalled carbon nanotubes (MWCNT) have elicited great interest in biomedical applications due to their extraordinary physical, chemical, and optical properties. Intravenous administration of MWCNT-based medical imaging agents and drugs in animal models was utilized. However, the potential harmful health effects of MWCNT administration in humans have not yet been elucidated. Furthermore, to date, there are no apparent reports regarding the precise mechanisms of translocation of MWCNT into target tissues and organs from blood circulation. This study demonstrates that exposure to MWCNT leads to an increase in cell permeability in human microvascular endothelial cells (HMVEC). The results obtained from this study also showed that the MWCNT-induced rise in endothelial permeability is mediated by reactive oxygen species (ROS) production and actin filament remodeling. In addition, it was found that MWCNT promoted cell migration in HMVEC. Mechanistically, MWCNT exposure elevated the levels of monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule 1 (ICAM-1) in HMVEC. Taken together, these results provide new insights into the bioreactivity of MWCNT, which may have implications in the biomedical application of MWCNT in vascular targeting, imaging, and drug delivery. The results generated from this study also elucidate the potential adverse effects of MWCNT exposure on humans at the cellular level.     

Noradrenaline-induced contraction mediated by endothelial COX-1 metabolites in the rat coronary artery

Noradrenaline-induced contraction of the rat coronary arteries was significantly augmented by the presence of NG-nitro-L-arginine methyl ester (L-NAME) and arachidonic acid. The experiments in the study presented here were undertaken to characterize pharmacologically the augmented noradrenaline-induced contraction in ring preparations of rat coronary arteries. The contraction was stopped by a chemical remover of endothelium (saponin). Oxygen radical scavengers, superoxide dismutase and catalase, significantly attenuated the contraction. Cyclooxygenase-1 inhibitors (flurbiprofen, 10(-7) M) attenuated the noradrenaline-induced contraction and cyclooxygenase-2 (nimesulide, 10(-7) M) slightly attenuated the contraction. A thromboxane A2 (TXA2) synthetase inhibitor (OKY-046) and a TXA2 receptor antagonist (S-1452) did not affect the contraction. Based on these results, it was suggested that the contraction induced by noradrenaline in the rat coronary artery in the presence of L-NAME and arachidonic acid is endothelium-dependent, and that it involves reactive oxygen species and endothelial cyclooxygenase-1 metabolites of arachidonic acid.     

Cell permeability, migration, and reactive oxygen species induced by multiwalled carbon nanotubes in human microvascular endothelial cells

Multiwalled carbon nanotubes (MWCNT) have elicited great interest in biomedical applications due to their extraordinary physical, chemical, and optical properties. Intravenous administration of MWCNT-based medical imaging agents and drugs in animal models was utilized. However, the potential harmful health effects of MWCNT administration in humans have not yet been elucidated. Furthermore, to date, there are no apparent reports regarding the precise mechanisms of translocation of MWCNT into target tissues and organs from blood circulation. This study demonstrates that exposure to MWCNT leads to an increase in cell permeability in human microvascular endothelial cells (HMVEC). The results obtained from this study also showed that the MWCNT-induced rise in endothelial permeability is mediated by reactive oxygen species (ROS) production and actin filament remodeling. In addition, it was found that MWCNT promoted cell migration in HMVEC. Mechanistically, MWCNT exposure elevated the levels of monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule 1 (ICAM-1) in HMVEC. Taken together, these results provide new insights into the bioreactivity of MWCNT, which may have implications in the biomedical application of MWCNT in vascular targeting, imaging, and drug delivery. The results generated from this study also elucidate the potential adverse effects of MWCNT exposure on humans at the cellular level.     

舒尼替尼经线粒体内活性氧引起心肌细胞凋亡

目的探讨舒尼替尼引起心肌细胞凋亡的作用机制。方法 H9C2细胞分别用舒尼替尼0.1,1和10μmol.L-1处理24,48,72 h,MTT法测定细胞存活率;流式细胞仪分别测定处理24 h细胞的凋亡、细胞内活性氧(ROS)、线粒体膜电位(ΔΨm)水平及胱天蛋白酶3活性。结果与同一时间点正常对照组相比,舒尼替尼1,10μmol.L-1处理24,48,72 h后,细胞存活率分别明显下降了22%和32%(24 h);41%和68%(48 h);62%和82%(72 h)(P<0.05)。与正常对照组相比,舒尼替尼1,10μmol.L-1作用24 h后,心肌细胞内ROS水平显著升高(4.41±0.76 vs 8.68±0.74,3.57±1.45)(P<0.05),线粒体膜电位下降(309±6 vs 244±28,174±2)(P<0.05),胱天蛋白酶3活性升高(0.96±0.13 vs 59.40±13.17,79.90±0.06)(P<0.05)及细胞凋亡率增加(〔6.03±0.40)%vs(21.05±5.55)%,(59.05±4.62)%〕(P<0.05)。结论舒尼替尼可通过诱导心肌细胞内ROS的产生,经线粒体内途径引起心肌细胞凋亡。     

活性氧类物质在化学物诱导的细胞凋亡中的调节作用(英文)

细胞凋亡是在生理或病理条件下 ,为维持内环境的稳定的一种程序性细胞死亡。诱导细胞凋亡的因素可分为物理性、化学性和生物性因素。很多实验数据表明氧化应激在细胞凋亡的发生过程中具有重要作用。各种活性氧类物质如超氧阴离子、过氧化氢、羟自由基和一氧化氮均与细胞凋亡的发生有关。但是活性氧类物质诱导凋亡发生的机理尚未阐明。本综述在对细胞凋亡和氧化应激描述的基础上着重讨论活性氧类物质诱导细胞凋亡发生的可能机理。在以后的研究中 ,将着重于探讨化学物质通过产生活性氧类物质而影响细胞凋亡的机理及建立相关的生物标志物。     

长春西汀对体外培养的人脐静脉内皮细胞脂质过氧化的影响

目的 观察长春西汀(吲哚类生物碱,治疗和预防缺血性脑血管病)对活性氧造成的人脐静脉内皮细胞(HUVEC)脂质过氧化损伤的影响.方法 用含100 g·L-1小牛血清的DMEM培养液体外培养HUVEC,用不同浓度(10、20、、50 100 μmol·L-1)的H2O2作为外源性活性氧,作用于HUVEC,用3H-胸腺嘧啶脱氧核苷掺入和细胞周期时相测定等方法,观察给予不同浓度(10、50、100 mmol·L-1)长春西汀预处理后,长春西汀对H2O2作用后的HUVEC增殖的影响.结果 H2O2对HUVEC的增殖活性具有剂量依赖性抑制作用;长春西汀可以抑制H2O2诱导的脂质过氧化损伤,且具有剂量依赖性.结论 长春西汀对动脉粥样硬化的防治可能具有应用前景.     

Effects of vasodilators on cyclic contraction induced by 3,4-diaminopyridine in isolated porcine coronary arteries

3,4-Diaminopyridine (3,4-DAP), which is known to decrease K conductance, produced spontaneous repetitive phasic contractions of a regular (28/60) and an irregular (15/60) cycle or tonic contraction (16/60) following a latent period of 5-100 min in isolated porcine coronary arteries. Effects of pinacidil, a newly-synthesized vasodilator, were investigated using the preparation in which 3,4-DAP produced phasic contractions of the regular cycle in comparison with those of various vasodilators. Pinacidil produced dose-dependent prolongation of the cycle and reduced the peak tension and the tension at the relaxation phase, a mode of action that closely resembles that of nicorandil, suggesting the increase in K conductance and hyperpolarization. Nifedipine (10(-8) M) and dilazep (10(-4) M) markedly reduced the peak tension, while adenosine, dipyridamole and nitroglycerin did not produce such effects. The latter three drugs produced a prolongation of the cycle and reduced the tension of the relaxation phase. These data suggest that reduction of K conductance and activation of the voltage-dependent Ca channel may play an important role in initiation of the spontaneous repetitive phasic contraction in porcine coronary artery.     

DNA damage induced by methylated trivalent arsenicals is mediated by reactive oxygen species

Arsenic is a human carcinogen; however, the mechanisms of arsenic's induction of carcinogenic effects have not been identified clearly. We have shown previously that monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)) are genotoxic and can damage supercoiled phiX174 DNA and the DNA in peripheral human lymphocytes in culture. These trivalent arsenicals are biomethylated forms of inorganic arsenic and have been detected in the urine of subjects exposed to arsenite and arsenate. We show here by molecular, chemical, and physical methods that reactive oxygen species (ROS) are intermediates in the DNA-damaging activities of MMA(III) and DMA(III). Using the phiX174 DNA nicking assay we found that the ROS inhibitors Tiron, melatonin, and the vitamin E analogue Trolox inhibited the DNA-nicking activities of both MMA(III) and DMA(III) at low micromolar concentrations. The spin trap agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) also was effective at preventing the DNA nicking induced by MMA(III) and DMA(III). ESR spectroscopy studies using DMPO identified a radical as a ROS intermediate in the DNA incubations with DMA(III). This radical adduct was assigned to the DMPO-hydroxyl free radical adduct on the basis of comparison of the observed hyperfine splitting constants and line widths with those reported in the literature. The formation of the DMPO-hydroxyl free radical adduct was dependent on time and the presence of DMA(III) and was completely inhibited by Tiron and Trolox and partially inhibited by DMSO. Using electrospray mass spectrometry, micromolar concentrations of DMA(V) were detected in the DNA incubation mixtures with DMA(III). These data are consistent with the conclusions that the DNA-damaging activity of DMA(III) is an indirect genotoxic effect mediated by ROS-formed concomitantly with the oxidation of DMA(III) to DMA(V).     

Differential inhibition by oxygen radicals of vasoactive amines-induced contractions in the porcine coronary artery

Reactive oxygen species (ROS) play an important role in normal metabolic and signaling processes. Excess ROS, however, can cause severe cardiovascular damage. Thus, the present study was designed to examine effects of H(2)O(2) and xanthine plus xanthin oxidase (X/XO) on the serotonin (5HT), histamine (His) and acetylcholine (ACh)-induced contractions of porcine coronary arteries. In addition, to explore the site of ROS formation and species of it, the inhibitory effects of edaravone and EDTA were also tested. 5HT- and His-induced contractions were suppressed by H(2)O(2) and X/XO treatment. However, these suppressions of ACh-induced contraction by H(2)O(2) treatment was relatively weak and X/XO treatment caused no suppression on ACh-induced contraction. In the presence of edaravone which is thought to be a scavenger for .OH, significant decrease of inhibition of 5HT- and His-induced contractions was observed when coronary artery strips were treated with X/XO, but not H(2)O(2). On the other hand, inhibitory effects by EDTA treatments were also observed in X/XO treatments. These results suggest that 1) ROSs produced by additions of H(2)O(2) or X/XO are considered to be responsible for several physiological functions of coronary artery contractions, 2) the site of ROS produced by X/XO system, probably .OH, was outside the cell, but the inhibitory action of H(2)O(2), was inside the cell, and 3) a low susceptibility of ACh-induced contraction to H(2)O(2) and X/XO may indicates the signal transduction pathway(s) of ACh-induced contraction is different from those of 5HT and His.     

Involvement of endothelial cyclo-oxygenase metabolites in noradrenaline-induced contraction of rat coronary artery

1. Noradrenaline (NA; 0.3 micromol/L) caused a contraction of the rat coronary artery that markedly increased in the presence of the nitric oxide synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME; 100 micromol/L) and arachidonic acid (1 micromol/L; P < 0.05). 2. The present experiments attempted to elucidate the endothelium dependency of the contraction and to pharmacologically characterize the factors involved in the contraction induced by NA (0.3 micromol/L) in the presence of L-NAME and arachidonic acid in ring preparations of the rat coronary artery. 3. The NA (0.3 micromol/L)-induced contraction was attenuated by a chemical remover of the endothelium (saponin at concentrations of 0.1 and 0.4 mg/mL) in a concentration-dependent manner (P < 0.05). 4. The cyclo-oxygenase (COX)-1 inhibitor flurbiprofen (0.01-1 micromol/L) and the COX-2 inhibitor nimesulide (0.01-1 micromol/L) attenuated the NA-induced contraction in a concentration-dependent manner and the inhibitory effect of flurbiprofen was significantly more potent than that of nimesulide (P < 0.05). The 5-lipoxigenase inhibitor ZM-230487 (1 micromol/L) did not affect the NA-induced contraction. 5. The thromboxane A2 (TXA2) synthetase inhibitor OKY-046 (30 micromol/L) and the TXA2 antagonist S-1452 (0.1-10 micromol/L) did not attenuate the NA-induced contraction. 6. These results indicate that the contraction induced by NA in the rat coronary artery in the presence of L-NAME and arachidonic acid is endothelium dependent and is due to endothelial COX metabolites of arachidonic acid.     

白藜芦醇诱导K562细胞凋亡过程中活性氧水平的改变

目的：探讨白藜芦醇对K562细胞的凋亡诱导效应和对K562细胞内活性氧水平的影响。方法：应用噻唑蓝(MTr)比色法、光镜、DNA琼脂糖凝胶电泳和流式细胞术(FCM)检测细胞凋亡；FCM测定细胞内活性氧(ROS)水平。结果：白藜芦醇显著抑制K562细胞增殖，并呈现典型的凋亡形态学改变；DNA电泳可见梯状条带出现；FCM分析显示S期细胞数明显增多，出现S／G，期阻滞，ROS水平升高。结论：白藜芦醇诱导K562细胞凋亡与细胞内活性氧水平升高有关。     

他巴唑对甲状腺细胞活性氧的影响

目的　观察他巴唑对甲状腺细胞活性氧 (ROS)的影响 ,探讨其作用的构效关系。方法　以 2′ ,7′二氯氢化荧光素和氢乙啡啶为细胞内H2 O2 与O- ·2 的荧光探针 ,以流式细胞术观察他巴唑及其结构类似物对甲状腺细胞活性氧的影响。结果　他巴唑对甲状腺细胞内外源性ROS均有清除作用 ,且这一作用呈剂量依赖性 ;无巯基的他巴唑结构类似物咪唑对ROS无清除作用 ,而谷胱甘肽过氧化物酶抑制物二乙马来酸可部分抑制他巴唑这种作用。结论　他巴唑对甲状腺细胞活性氧具清除效应 ,且这一效应可能与其分子中巯基有关     

Suppression of Fas ligand expression on endothelial cells by arsenite through reactive oxygen species

Exposure of rats to high doses of quartz and other insoluble isometric particles can produce lung tumors. In contrast, after exposure of such particles in hamsters no tumor outcome has been observed. Recent studies have demonstrated that the tumorigenic effect of particles is closely linked to the induction of inflammatory processes and the subsequent formation and persistence of mutagenic oxidative DNA-modifications. Species-specific differences in sensitivity to particles should therefore be reflected in the molecular reaction of the lung cells. We exposed rats and hamsters to two different doses of quartz (0.3 mg, 1.2 mg/100 g body weight) by intratracheal instillation and characterized the dose-related pattern of pulmonary inflammation (neutrophil recruitment, TNF), toxicity (protein content, surfactant phospholipids), antioxidant defence (glutathione content), mutagenicity (8-oxoguanine, p53) and proliferation. Our results clearly demonstrate a significantly higher response of the rat to quartz exposure for all determined molecular and cellular parameters. Therefore the examination of these parameters in humans would contribute to the evaluation of the relevance of rats or hamsters as models to predict particle-induced human lung cancer risk.     

Generation of reactive oxygen species induced by gold nanoparticles under x-ray and UV Irradiations

The radiosensitizing effect of 5--250 nm diameter Au nanoparticles (AuNPs) in water was investigated under irradiations of diagnostic x-ray and UV light. Enhanced generations of hydroxyl radical (OH) and superoxide anion (O₂⁻) were confirmed from their dependencies on the absorbed energy, ethanol concentration and AuNPs' concentration. Two kinds of fluorescent probes revealed that the reactive oxygen species (ROS) generation rate under x-ray irradiation was enhanced by factors of 1.46 for OH and 7.68 for O₂⁻. Photo- and Auger electron charge transfer is possibly relevant to generation of O₂⁻ near the particle surface, whereas fluorescent x-rays are involved in generation of OH in the secondary water radiolysis. Smaller diameter AuNPs with larger surface area showed a greater yield of ROS. An inverse proportion of ROS generation to the AuNPs' diameter suggests a catalytic function of AuNPs' surface for enhanced ROS generation.

From the Clinical Editor

This paper investigates the effects of UV and X-ray irradiation on reactive oxygen species induction of gold nanoparticles, concluding that smaller diameter AuNPs with larger surface area lead to a greater yield of ROS probably due to catalytic effects. The paper may be important for the development of novel non-toxic radiation sensitizers.