Reactive oxygen species and ischemic cerebrovascular disease

Stroke is an emerging major health problem often resulting in death or disability. Hyperlipidemia, high blood pressure and diabetes are well established risk factors. Endothelial dysfunction associated with these risk factors underlies pathological processes leading to atherogenesis and cerebral ischemic injury. While mechanisms of disease are complex, endothelial dysfunction involves decreased nitric oxide (NO) and elevated levels of reactive oxygen species (ROS). At physiological levels, ROS participate in regulation of cellular metabolism. However, when ROS increase to toxic levels through imbalance of production and neutralization by antioxidant enzymes, they cause cellular injury in the form of lipid peroxidation, protein oxidation and DNA damage. Central nervous system cells are more vulnerable to ROS toxicity due to their inherent higher oxidative metabolism and less antioxidant enzymes, as well as higher content of membranous fatty acids. During ischemic stroke, ROS concentration rises from normal low levels to a peak point during reperfusion possibly underlying apoptosis or cellular necrosis. Clinical trials and animal studies have shown that natural compounds can reduce oxidative stress due to excessive ROS through their antioxidant properties. With further study, we may be able to incorporate these compounds into clinical use with potential efficacy for both the treatment and prevention of stroke.     

Low concentration of oxidant and nitric oxide donors stimulate proliferation of human endothelial cells in vitro

Proliferation of human umbilical vein endothelial cells in vitro was inhibited by high concentrations of oxidants and nitric oxide donors but stimulated by low (micromolar or submicromolar) concentrations of hydrogen peroxide, menadione, tert-butyl hydroperoxide, AAPH, nitroglycerin, SIN-1 and sodium nitroprusside. The stimulation seems to be dependent upon generation of secondary reactive oxygen species as inferred from attenuation of cell proliferation by superoxide dismutase and catalase. These results point to another type of possible artefact of cell culture, viz. stimulation of cell proliferation by low concentrations of oxidants.     

Nitric oxide and ABA in the control of plant function

Abscisic acid (ABA) and nitric oxide (NO) are both extremely important signalling molecules employed by plants to control many aspects of physiology. ABA has been extensively studied in the mechanisms which control stomatal movement as well as in seed dormancy and germination and plant development. The addition of either ABA or NO to plant cells is known to instigate the actions of many signal transduction components. Both may have an influence on the phosphorylation of proteins in cells mediated by effects on protein kinases and phosphatases, as well as recruiting a wide range of other signal transduction molecules to mediate the final effects. Both ABA and NO may also lead to the regulation of gene expression. However, it is becoming more apparent that NO may be acting downstream of ABA, with such action being mediated by reactive oxygen species such as hydrogen peroxide in some cases. However not all ABA responses require the action of NO. Here, examples of where ABA and NO have been put together into the same signal transduction pathways are discussed.     

Growth hormone induces eNOS expression and nitric oxide release in a cultured human endothelial cell line

Growth hormone deficiency is linked to cardiovascular disease and particularly increased peripheral vascular resistance. Surprisingly, its role in endothelial nitric oxide (NO) synthetase (eNOS) regulation and NO release is basically unknown. We therefore studied the effects of different doses of somatotropin in cultures of a human endothelial cell line (EAhy926). We investigated expression and activity of eNOS, as well as other target genes known to be deregulated in cardiovascular disease including E-selectin and the lectin-like oxidized low density lipoprotein receptor. Treatment of cultured human endothelial cells with somatotropin resulted in significant (P<0.05) increases of eNOS gene and protein expression, as well as NO release, whereas production of intracellular reactive oxygen species was significantly reduced, at the highest somatotropin dose level. The enhanced eNOS gene/protein expression and enzyme activity correlate well. Our findings are suggestive for a novel role of growth hormone in endothelial biology, and particularly NO production.     

Resveratrol prevents hyperglycemia-induced endothelial dysfunction via activation of adenosine monophosphate-activated protein kinase

Endothelial dysfunction secondary to persistent hyperglycemia plays a key role in the development of type 2 diabetic vascular disease. The aim of the present study was to examine the protective effect of resveratrol against hyperglycemia-induced endothelial dysfunction. In cultured human umbilical vein endothelial cells (HUVECs), resveratrol (10-100 μM) concentration dependently enhanced phosphorylation of endothelial nitric oxide synthesis (eNOS) at Ser1177 and nitric oxide (NO) production. In addition, resveratrol can increase the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) at Thr172 and suppress high glucose-induced generation of superoxide anion. In mouse aortic rings, resveratrol (1-100 μM) elicited endothelium-dependent vasodilatations and alleviated high glucose-mediated endothelial dysfunction. All these beneficial effects of resveratrol on the endothelium were abolished by pharmacological antagonism of AMPK by compound C. These results provide new insight into the protective properties of resveratrol against endothelial dysfunction caused by high glucose, which is attributed to the AMPK mediated reduction of superoxide level.     

Nitric oxide counteracts cytotoxic processes mediated by reactive oxygen species in plant tissues

Many environmental conditions subject plants to oxidative stress, in which reactive oxygen species (ROS) are overproduced. These ROS act as transduction signals in plant defense responses, but also cause effects that result in cellular damage. Since nitric oxide (NO) is a bioactive molecule able to scavenge ROS, we analyzed its effect on some cytotoxic processes produced by ROS in potato (Solanum tuberosum L. cv. Pampeana) leaves. Two NO donors: (i) sodium nitroprusside and (ii) a mixed solution of ascorbic acid and NaNO₂, were able to prevent chlorophyll loss mediated by the methyl viologen herbicide diquat (a ROS generator), with effective concentrations falling between 10 and 100 μM of the donors. This protection was mimicked by thiourea and penicillamine, two antioxidant compounds. Residual products from NO generation and decomposition failed to prevent chlorophyll decline. A specific NO scavenger, the potassium salt of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), arrested NO-mediated chlorophyll protection. In addition, some events mediated by ROS during infection of potato leaves with Phytophthora infestans (race 1, 4, 7, 8, 10, 11, mating type A2) were also examined. In this sense, NO proved to markedly decrease ion leakage and the number of lesions, indicative of cell death, produced upon infection in potato leaves. The NO-mediated decrease in ion leakage was also inhibited by carboxy-PTIO. Fragmentation of DNA diminished when P. infestans-infected potato leaves were treated with 100 μM SNP. These results suggest that, acting as an antioxidant, NO can strongly counteract many ROS-mediated cytotoxic processes in plants. Moreover, the evidence of NO functionality in the plant kingdom is strengthened by this work. Received: 18 December 1998 / Accepted: 19 January 1999     

NO对镉胁迫下小麦根系生长发育的生理影响

为了探究外源物一氧化氮（NO）供体硝普钠（sodium nitroprusside,SNP）对Cd²⁺胁迫下小麦根系生长发育和活性氧代谢的影响,以小麦（Triticum aestivum L.）为材料,研究10 mmol/L CdCl₂胁迫下,30 μmol/L硝普钠（含一氧化氮NO）对小麦根系生长发育和活性氧代谢的影响。结果显示,外施SNP后,Cd²⁺胁迫下的小麦根长度、鲜重与干重较单独镉胁迫处理分别上升了48.0%、107.7%和87.3%,根系超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）、抗坏血酸过氧化物酶（APX）的活性分别上升了28.5%、7.4%、19.2%和9.8%,根中超氧自由基（O₂^.-）和过氧化氢（H₂O₂）的含量分别降低了80.5％和47.0％;同时外施SNP,使镉胁迫下小麦根中的可溶性糖含量和脯氨酸含量分别上升了24.7％和22.1%;使根中丙二醛（MDA）含量降低了30.2％;使根系活力上升了15.3%。因此,外源NO在一定程度上可以显著提高小麦根的抗氧化能力,增强小麦的抗逆性,缓解镉对小麦根系的毒害,进而促进小麦幼苗根系的生长发育。     

Nitric oxide mediates tightening of the endothelial barrier by ascorbic acid

Vitamin C, or ascorbic acid, decreases paracellular endothelial permeability in a process that requires rearrangement of the actin cytoskeleton. To define the proximal mechanism of this effect, we tested whether it might involve enhanced generation and/or sparing of nitric oxide (NO) by the vitamin. EA.hy926 endothelial cells cultured on semi-porous filter supports showed decreased endothelial barrier permeability to radiolabeled inulin in response to exogenous NO provided by the NO donor spermine NONOATE, as well as to activation of the downstream NO pathway by 8-bromo-cyclic GMP, a cell-penetrant cyclic GMP analog. Inhibition of endothelial nitric oxide synthase (eNOS) with N_ω-nitro-l-arginine methyl ester increased endothelial permeability, indicating a role constitutive NO generation by eNOS in maintaining the permeability barrier. Inhibition of guanylate cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one also increased endothelial permeability and blocked barrier tightening by spermine NONOATE. Loading cells with what are likely physiologic concentrations of ascorbate decreased endothelial permeability. This effect was blocked by inhibition of either eNOS or guanylate cyclase, suggesting that it involved generation of NO by eNOS and subsequent NO-dependent activation of guanylate cyclase. These results show that endothelial permeability barrier function depends on constitutive generation of NO and that ascorbate-dependent tightening of this barrier involves maintaining NO through the eNOS/guanylate cyclase pathway.     

The enhancing action of D-galactosamine on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS.     

Antioxidants Inhibit Angiogenesis In Vivo through Down-regulation of Nitric Oxide Synthase Expression and Activity

Although reactive oxygen species (ROS) participate in many cellular mechanisms, only few data exist concerning their involvement in physiological angiogenesis. The aim of the present work was to elucidate possible mechanisms through which ROS affect angiogenesis in vivo, using the model of the chicken embryo chorioallantoic membrane (CAM). Superoxide dismutase (SOD) and its membrane permeable mimetic tempol, dose dependently decreased angiogenesis and down-regulated inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. The NADPH oxidase inhibitors, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and apocynin, but not allopurinol, also had a dose dependent inhibitory effect on angiogenesis and NO production in vivo. Catalase and the intracellular hydrogen peroxide (H²O²) scavenger sodium pyruvate decreased, while H²O² increased in a dose-dependent manner the number of CAM blood vessels, as well as the expression and activity of iNOS. Dexamethasone, which down-regulated NO production by iNOS and l-NAME, but not d-NAME, dose dependently decreased angiogenesis in vivo. These data suggest that antioxidants affect physiological angiogenesis in vivo, through regulation of NOS expression and activity.     

Interplay between ferritin metabolism, reactive oxygen species and nitric oxide

 The biological relevance of each of the three inorganic species – iron, oxygen, and nitric oxide (NO) – is crucial. Moreover, their metabolic pathways cross each other and thus create a complex network of connections responsible for the regulation of many essential biological processes. The iron storage protein ferritin, one of the main regulators of iron homeostasis, influences oxygen and NO metabolism. Here, examples are given of the biological interactions of the ferritin molecule (ferritin iron and ferritin shell) with reactive oxygen species (ROS) and NO. The focus is the regulation of ferritin expression by ROS and NO. From these data, ferritin emerges as an important cytoprotective component of the cellular response to ROS and NO. Also, by its ability to alter the amount of intracellular "free" iron, ferritin may affect the metabolism of ROS and NO. It is proposed that this putative activity of ferritin may constitute a missing link in the regulatory loop between iron, ROS, and NO. Received: 2 January 1997 / Accepted: 9 June 1997     

Nitric oxide reduces Cu toxicity and Cu-induced NH4+ accumulation in rice leaves

Nitric oxide (NO) is a highly reactive, membrane-permeable free radical, which has recently emerged as an important antioxidant. Here we investigated the protective effect of NO against the toxicity and NH₄⁺ accumulation in rice leaves caused by excess CuSO₄ (10 mmol L⁻¹). It was found that free radical scavengers (sodium benzoate, thiourea, and reduced glutathione) reduced the toxicity and NH₄⁺ accumulation in rice leaves caused by excess CuSO₄. NO donor sodium nitroprusside (SNP) was also effective in reducing CuSO₄-induced toxicity and NH₄⁺ accumulation in rice leaves. The protective effect of SNP on the toxicity and NH₄⁺ accumulation can be reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl- imidazoline-1-oxyl-3-oxide, a NO scavenger, suggesting that the protective effect of SNP is attributable to NO released. Results obtained in the present study suggest that reduction of CuSO₄-induced toxicity and NH₄⁺ accumulation by SNP is most likely mediated through its ability to scavenge active oxygen species.     

Nitric oxide stimulates seed germination and counteracts the inhibitory effect of heavy metals and salinity on root growth of Lupinus luteus

Nitric oxide (NO) is a bioactive molecule, which in plants was found to function as prooxidant as well as antioxidant. In the present study, we found that NO donor sodium nitroprusside (SNP) stimulates seed germination and root growth of lupin (Lupinus luteus L. cv. Ventus). Seed germination is promoted at concentrations between 0.1 and 800 μM SNP in a dose-dependent manner. The stimulation was most pronounced after 18 and 24 h and ceased after 48 h of imbibition. The promoting effect of NO on seed germination persisted even in the presence of heavy metals (Pb, Cd) and sodium chloride. Pretreatment of lupin seedlings for 24 h with 10 μM SNP resulted in efficient reduction of the detrimental effect of the abiotic stressors on root growth and morphology. The inhibitory effect of heavy metals on root growth was accompanied by increased activity of superoxide dismutase (SOD, EC 1.15.1.1.), which in roots preincubated with SNP was significantly higher. Some changes in the activity of other antioxidant enzymes, peroxidase (POX, EC 1.11.1.7) and catalase (CAT, EC 1.11.1.6) were also detected. Using the superoxide anion (O₂^•–)-specific indicator dihydroethidium (DHE), we found intense DHE-derived fluorescence in heavy metal-stressed roots, whereas in those pretreated with SNP the fluorescence was very low, comparable to the level in unstressed roots. On the basis of the above data, we conclude that the protective effect of NO in stressed lupin roots may be at least partly due to the stimulation of SOD activity and/or direct scavenging of the superoxide anion.     

Therapeutic approaches using nitric oxide in infants and children

Pulmonary hypertension contributes significantly to the morbidity and mortality associated with many pediatric pulmonary and cardiac diseases. Nitric oxide, a gas molecule, is a unique pharmaceutical agent that can be inhaled and thus delivered directly to the lung. Inhaled nitric oxide was approved by the FDA in 1999 as a therapy for infants with persistent pulmonary hypertension. Since then, the use of inhaled nitric oxide has expanded to other neonatal and pediatric conditions, and our knowledge of its properties and mechanisms of action has increased tremendously. This review discusses the physiology of nitric oxide signaling, the most common indications for its clinical use, and promising new investigations that may enhance endogenous production of nitric oxide and/or improve vascular response to it.     

Insulin resistance,lipotoxicity and endothelial dysfunction

The number of people with the insulin-resistant conditions of type 2 diabetes mellitus (T2DM) and obesity has reached epidemic proportions worldwide. Eighty percent of people with T2DM will die from the complications of cardiovascular atherosclerosis. Insulin resistance is characterised by endothelial dysfunction, which is a pivotal step in the initiation/progression of atherosclerosis. A hallmark of endothelial dysfunction is an unfavourable imbalance between the bioavailability of the antiatherosclerotic signalling molecule nitric oxide (NO) and proatherosclerotic reactive oxygen species. In this review we discuss the mechanisms linking insulin resistance to endothelial dysfunction, with a particular emphasis on a potential role for a toxic effect of free fatty acids on endothelial cell homeostasis.     

Oxidized LDLs affect nitric oxide and radical generation in brain endothelial cells

There is an increase in the generation of reactive oxygen species and nitric oxide in the cerebral microcirculation in Alzheimer's disease. The factors that cause this increase in oxidative stress have not been identified. Increasing evidence suggests that there are common mechanisms in atherosclerosis and Alzheimer's disease. The objective of this study was to determine the effects of oxidized low density lipoproteins (LDLs) on brain endothelial cells. Cultured rat brain endothelial cells were treated with either native LDL (10 microg/ml) or LDL oxidized in vitro using 4-hydroxy-2-nonenal (HNE-LDL) (10 microg/ml), for 24h. The results showed that HNE-LDL significantly increased production of nitric oxide (p<0.01), decreased membrane fluidity (p<0.05), and increased reactive oxygen species generation (p<0.01). These data demonstrate that oxidized LDLs affect nitric oxide and radical generation in brain endothelial cells and could contribute to cerebrovascular dysfunction in Alzheimer's disease.     

Nitric oxide/cGMP protects endothelial cells from hypoxia-mediated leakiness

Leakiness of the endothelial bed is attributed to the over-perfusion of the pulmonary bed, which leads to high altitude pulmonary edema (HAPE). Inhalation of nitric oxide has been successfully employed to treat HAPE patients. We hypothesize that nitric oxide intervenes in the permeability of the pulmonary macrovascular endothelial bed to rectify the leaky bed under hypoxia. Our present work explores the underlying mechanism of 'hypoxia-mediated' endothelial malfunction by using human umbilical cord-derived immortalized endothelial cells, ECV-304, and bovine pulmonary artery primary endothelial cells. The leakiness of the endothelial monolayer was increased by two-fold under hypoxia in comparison to cells under normoxia, while optical tweezers-based tethering assays reported a higher membrane tension of endothelial cells under hypoxia. Phalloidin staining demonstrated depolymerization of F-actin stress fibers and highly polarized F-actin patterns in endothelial cells under hypoxia. Nitric oxide, 8-Br-cGMP and sildenafil citrate (phosphodiesterase type 5 inhibitor) led to recovery from hypoxia-induced leakiness of the endothelial monolayers. Results of the present study also suggest that 'hypoxia-induced' cytoskeletal rearrangements and membrane leakiness are associated with the low nitric oxide availability under hypoxia. We conclude that nitric oxide-based recovery of hypoxia-induced leakiness of endothelial cells is a cyclic guanosine monophosphate (cGMP)-dependent phenomenon.     

Nitric oxide-induced eosinophil apoptosis is dependent on mitochondrial permeability transition (mPT), JNK and oxidative stress: apoptosis is preceded but not mediated by early mPT-dependent JNK activation

Background

Eosinophils are critically involved in the pathogenesis of asthma. Nitric oxide (NO) is produced in high amounts in asthmatic lungs and has an important role as a regulator of lung inflammation. NO was previously shown to induce eosinophil apoptosis mediated via c-jun N-terminal kinase (JNK) and caspases. Our aim was to clarify the cascade of events leading to NO-induced apoptosis in granulocyte macrophage-colony stimulating factor (GM-CSF)-treated human eosinophils concentrating on the role of mitochondria, reactive oxygen species (ROS) and JNK.

Methods

Apoptosis was determined by flow cytometric analysis of relative DNA content, by Annexin-V labelling and/or morphological analysis. Immunoblotting was used to study phospho-JNK (pJNK) expression. Mitochondrial membrane potential was assessed by JC-1-staining and mitochondrial permeability transition (mPT) by loading cells with calcein acetoxymethyl ester (AM) and CoCl₂ after which flow cytometric analysis was conducted. Statistical significance was calculated by repeated measures analysis of variance (ANOVA) or paired t-test.

Results

NO-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) induced late apoptosis in GM-CSF-treated eosinophils. SNAP-induced apoptosis was suppressed by inhibitor of mPT bongkrekic acid (BA), inhibitor of JNK SP600125 and superoxide dismutase-mimetic AEOL 10150. Treatment with SNAP led to late loss of mitochondrial membrane potential. Additionally, we found that SNAP induces early partial mPT (1 h) that was followed by a strong increase in pJNK levels (2 h). Both events were prevented by BA. However, these events were not related to apoptosis because SNAP-induced apoptosis was prevented as efficiently when BA was added 16 h after SNAP. In addition to the early and strong rise, pJNK levels were less prominently increased at 20–30 h.

Conclusions

Here we demonstrated that NO-induced eosinophil apoptosis is mediated via ROS, JNK and late mPT. Additionally, our results suggest that NO induces early transient mPT (flickerings) that leads to JNK activation but is not significant for apoptosis. Thereby, we showed some interesting early events in NO-stimulated eosinophils that may take place even if the threshold for irreversible mPT and apoptosis is not crossed. This study also revealed a previously unknown physiological function for transient mPT by showing that it may function as initiator of non-apoptotic JNK signalling.