In vivo key role of reactive oxygen species and NHE-1 activation in determining excessive cardiac hypertrophy

Growing in vitro evidence suggests NHE-1, a known target for reactive oxygen species (ROS), as a key mediator in cardiac hypertrophy (CH). Moreover, NHE-1 inhibition was shown effective in preventing CH and failure; so has been the case for AT1 receptor (AT1R) blockers. Previous experiments indicate that myocardial stretch promotes angiotensin II release and post-translational NHE-1 activation; however, in vivo data supporting this mechanism and its long-term consequences are scanty. In this work, we thought of providing in vivo evidence linking AT1R with ROS and NHE-1 activation in mediating CH. CH was induced in mice by TAC. A group of animals was treated with the AT1R blocker losartan. Cardiac contractility was assessed by echocardiography and pressure-volume loop hemodynamics. After 7?weeks, TAC increased left ventricular (LV) mass by ~45% vs. sham and deteriorated LV systolic function. CH was accompanied by activation of the redox-sensitive kinase p90(RSK) with the consequent increase in NHE-1 phosphorylation. Losartan prevented p90(RSK) and NHE-1 phosphorylation, ameliorated CH and restored cardiac function despite decreased LV wall thickness and similar LV systolic pressures and diastolic dimensions (increased LV wall stress). In conclusion, AT1R blockade prevented excessive oxidative stress, p90(RSK) and NHE-1 phosphorylation, and decreased CH independently of hemodynamic changes. In addition, cardiac performance improved despite a higher work load.     

Regulation of hypertrophic and apoptotic signaling pathways by reactive oxygen species in cardiac myocytes

Increasing evidence suggests that oxidative and nitrosative stress play an important role in regulation of cardiac myocyte growth and survival. The cardiovascular system is continuously exposed to both reactive oxygen species (ROS) and nitrogen species (RNS), collectively termed reactive inflammatory species (RIS), and imbalances between the enzymes that regulate their bioavailability are associated with cardiac hypertrophy and the pathogenesis of cardiomyopathies, myocardial infarction and heart failure. It is now clear that RIS act as critical regulators of cardiac myocyte hypertrophy and apoptosis through control of redox-sensitive signaling cascades, such as tyrosine kinases and phosphatases, protein kinase C, and mitogen-activated protein kinases. This review will focus on the mechanisms by which ROS/RNS modulate cardiac myocyte growth and apoptosis induced by neurohormones and cytokines, and will discuss evidence for a role in the pathophysiology of heart failure.     

内源性H2S抑制angiotensin II引起的神经元活性氧水平的升高*

 目的：探讨内源性硫化氢（H2S）对血管紧张素II（Ang II）引起的延髓神经元活性氧（ROS）水平升高的作用及其可能机制。方法：首先培养原代延髓神经元;免疫荧光双标法鉴定神经元及内源性H2S生成酶胱硫醚β-合成酶（CBS）在神经元的表达;同时或单独给予Ang II （1 μmol/L）和丁酸钠（NaBu,一种CBS激动剂;100 μmol/L、250 μmol/L和500 μmol/L）,二氢乙啶荧光探针法测定ROS水平;采用总超氧化物歧化酶（SOD）活性检测试剂盒观察总SOD的活性;real-time PCR观察CBS mRNA的表达。结果：（1）原代培养的90% 以上的细胞为神经元,Ang II（1 μmol/L）升高延髓神经元ROS水平;（2）Ang II抑制神经元总SOD的活性;（3）荧光双标显示CBS在延髓神经元有表达,Ang II可降低CBS mRNA的表达;（4）NaBu（250 μmol/L和500 μmol/L）显著抑制Ang II引起的ROS水平的升高,且呈剂量依赖性效应。而NaBu单独对延髓神经元ROS水平作用不明显。结论：Ang II引起的延髓神经元ROS水平升高至少部分是通过降低总SOD的活性和CBS mRNA的表达而实现的;而内源性H2S可能通过相反的作用抑制这一过程。     

NADPH oxidase-dependent formation of reactive oxygen species contributes to angiotensin II-induced epithelial-mesenchymal transition in rat peritoneal mesothelial cells

The objective of the present study was to investigate the role of NADPH oxidase-dependent formation of reactive oxygen species (ROS) in the angiotensin II (Ang II)-induced epithelial-mesenchymal transition (EMT) and in the accumulation of extracellular matrix (ECM) in rat peritoneal mesothelial cells (RPMCs). Primary cultured RPMCs were incubated with serum-free media for 24 h in order to arrest and synchronize cell growth. The cells were treated with Ang II (10?? M) up to 48 h. Cells were pretreated with an Ang II type I receptor antagonist (losartan, 10?? M), or an inhibitor of NADPH, oxidase diphenyleneiodonium (DPI) (10?? M), for 1 h before addition of Ang II. The dichlorofluorescein (DCF)-sensitive cellular ROS were measured by fluorometric assay and confocal microscopy. RT-PCR was employed to detect the mRNA expression for the NADPH oxidase subunit p47phox, plasminogen activator inhibitor-1 (PAI-1), α-smooth muscle actin (α-SMA) and E-cadherin. PAI-1, α-SMA and p47phox protein expression were examined by Western blot analysis. Ang II significantly induced the production of intracellular ROS. DPI and losartan inhibited Ang II-induced ROS generation by 86.8% and 77.4% (p<0.05), respectively. Ang II significantly stimulated NADPH oxidase subunit p47phox mRNA and protein expression in RPMCs. Both losartan and DPI inhibited Ang II-induced up-regulation of p47phox mRNA by 37.3% and 67.8% (p<0.05), respectively. Ang II also stimulated α-SMA mRNA and protein expression and down-regulated E-cadherin mRNA expression in RPMCs. This effect was suppressed by both losartan and DPI. Ang II significantly up-regulated PAI-1 mRNA and protein expression and these were significantly suppressed by both losartan and DPI. In conclusion, NADPH oxidase-dependent formation of ROS mediates Ang II dependent EMT and accumulation of ECM in rat peritoneal mesothelial cells. NADPH oxidase may represent a potential therapeutic target in the management of peritoneal fibrosis.     

Protection from angiotensin II-induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats

Angiotensin converting enzyme 2 (ACE2), a newly discovered member of the renin-angiotensin system (RAS), is a potential therapeutic target for the control of cardiovascular disease owing to its key role in the formation of vasoprotective peptides from angiotensin II. The aim of the present study was to evaluate whether overexpression of ACE2 could protect the heart from angiotensin II-induced hypertrophy and fibrosis. Lentiviral vector encoding mouse ACE2 (lenti-mACE2) or GFP was injected intracardially in 5-day-old Sprague-Dawley rats. This resulted in expression of mACE2 in cardiac tissue for the duration of the study. Infusion of 200 ng kg-1 min-1 angiotensin II for 4 weeks resulted in an 80 mmHg increase in systolic blood pressure, a significant increase in the heart weight to body weight ratio (HW:BW), and marked myocardial fibrosis in control rats. Transduction with lenti-mACE2 resulted in significant attenuation of the increased HW:BW and myocardial fibrosis induced by angiotensin II infusion. These observations demonstrate that ACE2 overexpression results in protective effects on angiotensin II-induced cardiac hypertrophy and fibrosis.     

Serum amyloid A mediates human neutrophil production of reactive oxygen species through a receptor independent of formyl peptide receptor like-1

Serum amyloid A (SAA) is one of the acute-phase reactants, a group of plasma proteins that increases immensely in concentration during microbial infections and inflammatory conditions, and a close relationship between SAA levels and disease activity in rheumatoid arthritis (RA) has been observed. RA is an inflammatory disease, where neutrophils play important roles, and SAA is thought to participate in the inflammatory reaction by being a neutrophil chemoattractant and inducer of proinflammatory cytokines. The biological effects of SAA are reportedly mediated mainly through formyl peptide receptor like-1 (FPRL1), a G protein-coupled receptor (GPCR) belonging to the formyl peptide receptor family. Here, we confirmed the affinity of SAA for FPRL1 by showing that stably transfected HL-60 cells expressing FPRL1 were activated by SAA and that the response was inhibited by the use of the FPRL1-specific antagonist WRWWWW (WRW4). We also show that SAA activates the neutrophil NADPH-oxidase and that a reserve pool of receptors is present in storage organelles mobilized by priming agents such as TNF-alpha and LPS from Gram-negative bacteria. The induced activity was inhibited by pertussis toxin, indicating the involvement of a GPCR. However, based on FPRL1-specific desensitization and use of FPRL1 antagonist WRW4, we found the SAA-mediated effects in neutrophils to be independent of FPRL1. Based on these findings, we conclude that SAA signaling in neutrophils is mediated through a GPCR, distinct from FPRL1. Future identification and characterization of the SAA receptor could lead to development of novel, therapeutic targets for treatment of RA.     

Activation of tyrosine kinases by reactive oxygen species in vascular smooth muscle cells: significance and involvement of EGF receptor transactivation by angiotensin II

Enhanced production of reactive oxygen species (ROS) such as H(2)O(2) and a failure in ROS removal by scavenging systems are hallmarks of several cardiovascular diseases such as atherosclerosis and hypertension. ROS act as second messengers that play a prominent role in intracellular signaling and cellular function. In vascular smooth muscle cells (VSMCs), a vascular pathogen, angiotensin II, appears to initiate growth-promoting signal transduction through ROS-sensitive tyrosine kinases. However, the precise mechanisms by which tyrosine kinases are activated by ROS remain unclear. In this review, the current knowledge that suggests how certain tyrosine kinases are activated by ROS, along with their functional significance in VSMCs, will be discussed. Recent findings suggest that transactivation of the epidermal growth factor receptor by ROS requires metalloprotease-dependent heparin-binding epidermal growth factor-like growth factor production, whereas other ROS-sensitive tyrosine kinases such as PYK2, JAK2, and platelet-derived growth factor receptor require activation of protein kinase C-delta. Each of these ROS-sensitive kinases could mediate specific signaling critical for pathophysiological responses. Detailed analysis of the mechanism of cross-talk and the downstream function of these various tyrosine kinases will yield new therapeutic interventions for cardiovascular disease.     

Curcumin induces heme oxygenase 1 through generation of reactive oxygen species, p38 activation and phosphatase inhibition

Curcumin is a naturally occurring compound which is known to induce heme oxygenase 1 (HO-1), although the underlying mechanism has not been fully elucidated. This study investigates in detail the mechanism of HO-1 induction by curcumin in human hepatoma cells. There was increasing toxicity of curcumin at concentrations higher than 10 microM. Curcumin was found to induce HO-1 at doses of 10 to 25 microM. At both non-toxic and toxic doses, HO-1 induction was found to correlate with production of reactive oxygen species (ROS), suggesting a causative relationship. This was reinforced by the finding that pretreatment with the antioxidants N-acetylcysteine, vitamin E and catalase prevented HO-1 induction by curcumin. ROS production appeared to be mitochondrial in origin, and curcumin treatment resulted in depolarisation of the mitochondrial membrane potential. Nrf2 was induced by curcumin treatment, which was also partly ROS dependent. Using siRNA, Nrf2 was demonstrated to contribute to HO-1 induction. A panel of kinase inhibitors was used to examine the contribution of MAP kinases to the induction of HO-1 by curcumin. PKC and p38 MAPK activity are required for full induction of HO-1. Furthermore, curcumin also inhibited protein phosphatase activity. In conclusion, curcumin treatment results in ROS generation, activation of Nrf2 and MAP kinases and the inhibition of phosphatase activity in hepatocytes, and when curcumin is not administered in toxic doses, these multiple pathways converge to induce HO-1.     

Role of Rac-GTPase and reactive oxygen species in cardiac differentiation of stem cells

In the life of a cell, there is a constant balance between generation of reactive oxygen species (ROS) and activity of antioxidant defense mechanisms. Besides the damaging effects of ROS on many biomolecules, ROS also play a significant role in signal transduction pathways of growth factors suggesting a role of oxidative species in cell differentiation. ROS have recently been involved in the process of cardiac differentiation of stem cells. Several molecular mechanisms, including ones mediated by the GTPase Rac that underlie the regulatory role of ROS in the process of stem cell differentiation toward a cardiac lineage, are reviewed.     

Evidence that 4-aminobiphenyl, benzidine, and benzidine congeners produce genotoxicity through reactive oxygen species

4-Aminobyphenyl (4-Ab), benzidine (Bz), and Bz congeners were evaluated for their ability to induce genotoxicity through an oxidative mechanism. The mutagenicity of these compounds was tested in the presence and absence of Aroclor 1254-induced rat S9 mix using Salmonella typhimurium tester strain TA102, which is sensitive to agents producing reactive oxygen species (ROS). In the presence of S9, 4-Ab, Bz, N-acetyl-benzidine, and 3,3-dimethoxybenzidine were strongly mutagenic in TA102, whereas, 3,3,5,5-tetra-methylbenzidine, 3,3-dimethylbenzidine (O-tolidine), and N,N-diacetylbenzidine were not mutagenic. In addition, 3,3-dichlorobenzidine and 4,4-dinitro-2-biphenylamine were directly mutagenic in TA102. Incorporation of the free radical and metal scavengers, catalase, superoxide dismutase (SOD), butylated hydroxytolune (BHT), and ethylenediamine tetraacetic acid (EDTA) reduced the mutagenic responses of 4-Ab and Bz, whereas heat-inactivated catalase and SOD had no effect. 4-Ab and Bz also induced lipid peroxidation in the presence of S9 mix as shown using the thiobarbituric acid reactive substances assay. The results of this study indicate that 4-Ab and Bz induce mutations through the induction of ROS.     

Relation of Cardiotrophin-1 (CT-1) and cardiac transcription factor GATA4 expression in rat's cardiac myocytes hypertrophy and apoptosis

The aim of this study was to evaluate the role of GATA4 in hypertrophy and survival functions of CT-1 in the heart, and to apply chemical inhibitor strategies to assess a possible interaction of signaling pathways involved in these processes. Expression of GATA4 was determined at the mRNA expression (polymerase chain reaction, PCR) and protein binding activity (electrophoretic mobility shift assays, EMSAs) levels. Myocardial apoptosis was detected using the in-situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Parthenolide (an inhibitor to STAT3) and U0126 (an MEK inhibitor to block ERK1/2) were used to assess a possible interaction of signaling pathways involved in the processes. The results showed that CT-1 had a significant role in the expression of GATA4 mRNA and binding activity of cardiomyocytes. The stronger expression of GATA4 mRNA stimulated by CT-1 was essentially mediated by STAT3, and was negatively regulated by ERK1/2. The intercross relationship between STAT3 and ERK1/2 might assist CT-1 in cardiac hypertrophy. At the same time, CT-1 had its effect on anti-apoptosis and survival of cardiac myocytes. During this process, GATA4 expression showed a negative relationship with myocardial apoptosis. Obviously, STAT3 cannot affect the anti-apoptotic process of CT-1, but obstruction of ERK signaling pathway can inhibit the CT-1 anti-apoptotic effect significantly. Our data suggest that CT-1 can affect the expression of GATA4 mRNA and the binding activity of cardiac myocytes. GATA4 plays an important role in the hypertrophic effect of CT-1, in which STAT3 plays a primary role. The regulation of CT-1 on the anti-apoptotic process is mediated partly by GATA4, and can be inhibited by obstructing the ERK signaling pathway.     

Modulation of intracellular reactive oxygen species level in chondrocytes by IGF-1, FGF, and TGF-beta1

Growth factors are important in the development, maintenance and repair of cartilage. The principal aim of this study was to test the capacity of three growth factors with established roles in cartilage, namely insulin-like growth factor (IGF)-1, fibroblast growth factor (FGF) and transforming growth factor (TGF)-beta 1, to alter intracellular reactive oxygen species (ROS) levels. Explants of articular cartilage from young, mature, and aged rats were pretreated with IGF-1, FGF, or TGF-beta 1 and intracellular ROS levels were quantified using the free radical sensing probe dihydrorhodamine 123 (DHR 123), confocal microscopy, and densitometric image analysis. Viability of chondrocytes following ROS stress and growth factor treatment was assessed using the live/dead cytotoxicity assay, and the activities of the antioxidant enzymes--catalase (CAT), total superoxide dismutase (SOD), and glutathione peroxidase (GPX)--were measured spectrophotometrically by decay of the substrate from the reaction mixture. The effect of IGF-1 on ROS levels in cultured human chondrocytes also was examined. In rat cartilage, FGF did not significantly affect ROS levels or antioxidant enzyme activity in any age group. TGF-beta1 significantly increased cellular ROS levels in mature and old cartilage whereas in marked contrast, IGF-1 significantly and age-dependently reduced ROS levels. IGF-1 also had a potent antioxidant effect on cultured human chondrocytes. Pretreatment of rat cartilage with IGF-1 significantly enhanced the activity of GPX, without altering the activity of SOD or CAT, and protected chondrocytes against ROS-induced cell death. TGF-beta 1 had no significant effect on the activity of the antioxidant enzymes. Despite promoting ROS production, TGF-beta 1 was not cytotoxic. We concluded that TGF-beta 1 exhibits an acute pro-oxidant effect in cartilage that is not cytotoxic, suggesting a role in physiological cell signalling. In marked contrast, IGF-1 is a potent antioxidant in mature and aged rat and human chondrocytes, protecting cells against ROS-induced cell death probably through the enhancement of the activity of the antioxidant enzyme GPX.     

H2 S抑制Ang II引起的神经元活性氧水平升高的机制研究

目的: 探讨硫化氢(H₂S)对血管紧张素II(angiotensin II,Ang II)所致延髓神经元活性氧(reactive oxygen species,ROS)水平变化的影响,以及H₂S抗氧化应激的相关机制。方法: 采用原代培养的延髓神经元,分组如下:对照组、Ang II处理组、硫氢化钠(NaHS)处理组、NaHS+Ang II处理组和PD98059(p-ERK1/2蛋白的抑制剂)+ Ang II处理组。选用二氢乙啶(DHE)荧光探针法测定各组神经元胞内的ROS水平,Western blotting检测ERK1/2和p-ERK1/2蛋白的表达量,CCK-8法测定神经元的活性。结果: Ang II(终浓度为100 nmol/L)引起神经元ROS水平升高;NaHS(50~200 μmol/L)明显抑制Ang II引起的神经元ROS水平升高,但单独给予NaHS并不影响神经元ROS水平;p-ERK1/2蛋白的抑制剂PD98059也能明显抑制Ang II引起的神经元ROS水平的升高;适宜作用浓度的NaHS能够显著降低Ang II引起的神经元p-ERK1/2蛋白表达。结论: H₂S能够显著抑制Ang II引起的神经元ROS水平升高,其作用机制与MAPK家族中p-ERK1/2蛋白的表达有关。H₂S可能通过降低p-ERK1/2的表达量而产生抗氧化应激作用。     

The superoxide anion donor,potassium superoxide,induces pain and inflammation in mice through production of reactive oxygen species and cyclooxygenase-2

It is currently accepted that superoxide anion (O₂ ^•−) is an important mediator in pain and inflammation. The role of superoxide anion in pain and inflammation has been mainly determined indirectly by modulating its production and inactivation. Direct evidence using potassium superoxide (KO₂), a superoxide anion donor, demonstrated that it induced thermal hyperalgesia, as assessed by the Hargreaves method. However, it remains to be determined whether KO₂ is capable of inducing other inflammatory and nociceptive responses attributed to superoxide anion. Therefore, in the present study, we investigated the nociceptive and inflammatory effects of KO₂. The KO₂-induced inflammatory responses evaluated in mice were: mechanical hyperalgesia (electronic version of von Frey filaments), thermal hyperalgesia (hot plate), edema (caliper rule), myeloperoxidase activity (colorimetric assay), overt pain-like behaviors (flinches, time spent licking and writhing score), leukocyte recruitment, oxidative stress, and cyclooxygenase-2 mRNA expression (quantitative PCR). Administration of KO₂ induced mechanical hyperalgesia, thermal hyperalgesia, paw edema, leukocyte recruitment, the writhing response, paw flinching, and paw licking in a dose-dependent manner. KO₂ also induced time-dependent cyclooxygenase-2 mRNA expression in the paw skin. The nociceptive, inflammatory, and oxidative stress components of KO₂-induced responses were responsive to morphine (analgesic opioid), quercetin (antioxidant flavonoid), and/or celecoxib (anti-inflammatory cyclooxygenase-2 inhibitor) treatment. In conclusion, the well-established superoxide anion donor KO₂ is a valuable tool for studying the mechanisms and pharmacological susceptibilities of superoxide anion-triggered nociceptive and inflammatory responses ranging from mechanical and thermal hyperalgesia to overt pain-like behaviors, edema, and leukocyte recruitment.     

Neurokinin-1 receptor activation induces reactive oxygen species and epithelial damage in allergic airway inflammation

Background An induction of reactive oxygen species (ROS) is characteristic for inflammation but the exact pathways have not been identified for allergic airway diseases so far. Objective The aim of this study was to characterize the role of the tachykinin NK‐1 receptor on ROS production during allergen challenge and subsequent inflammation and remodelling. Methods Precision‐cut lung slices of ovalbumin (OVA)‐sensitized mice were cultivated and ROS‐generation in response to OVA challenge (10 μg/mL) was examined by the 2′,7′‐dichloroflourescein‐diacetate method. Long‐term ROS effects on epithelial proliferation were investigated by 5‐bromo‐2′‐deoxyuridine incorporation (72 h). In vivo, the results were validated in OVA‐sensitized animals which were treated intra‐nasally with either placebo, the tachykinin neurokinin 1 (NK‐1) receptor antagonist SR 140333 or the anti‐oxidant N‐acetylcystein (NAC) before allergen challenge. Inflammatory infiltration and remodelling were assessed 48 h after allergen challenge. Results ROS generation was increased by 3.7‐fold, which was inhibited by SR 140333. [Sar⁹,Met¹¹(O₂)]‐Substance P (5 nm ) caused a tachykinin NK‐1 receptor‐dependent fourfold increase in ROS generation. Epithelial proliferation was decreased by 68% by incubation with [Sar⁹,Met¹¹(O₂)]‐SP over 72 h. In‐vivo, treatment with SR 140333 and NAC reduced epithelial damage (91.4% and 76.8% vs. placebo, respectively, P<0.01) and goblet cell hyperplasia (67.4% and 50.1% vs. placebo, respectively, P<0.05), and decreased inflammatory cell influx (65.3% and 45.3% vs. placebo, respectively, P<0.01). Conclusion Allergen challenge induces ROS in a tachykinin NK‐1 receptor‐dependent manner. Inhibition of the tachykinin NK‐1 receptor reduces epithelial damage and subsequent remodelling in vivo. Therefore, patients may possibly benefit from treatment regime that includes radical scavengers or tachykinin NK‐1 receptor antagonists.     

Membrane fluidity, reactive oxygen species, and cell-mediated immunity: Implications in nutrition and disease

Membrane-associated functions are profoundly influenced by the fluidity and physical state of the membrane. These two features are in turn determined by the membrane lipid acyl chain profile. Reactive oxygen species (ROS) modify the acyl chains by lipid peroxidation of the unsaturated chains, thus affecting the fluidity and physical state of the membrane. By enhancing endogenous ROS levels, therefore, aging, and disease affect integral membrane function, such as the cell-mediated immune (CMI) reaction involving phagocyte membrane NADPH oxidase. This enzyme relies on triggering by membrane-inserted protein kinase C (PKC), for its superoxide (O2-) producing function. The molecular mechanism of the depressed immunocompetence in the aged, and in disease and malnutrition, may reside in the down-regulation of these two enzymes by excess ROS. These excess ROS arise from activated phagocytes in disease states, and from enhanced ROS from other sources in the aged, as well as from the decrease in antioxidants in the aged. Research should be intensified on PKC and NADPH oxidase function with the aim of unravelling the molecular mechanism of the depressed immunocompetence, and thence, of formulating appropriate intervention strategies against it.