Nitrosative stress inhibits production of the virulence factor alginate in mucoid Pseudomonas aeruginosa

Alginate is a critical virulence factor contributing to the poor clinical prognosis associated with the conversion of Pseudomonas aeruginosa to mucoid phenotypes in cystic fibrosis (CF). An important mechanism of action is its ability to scavenge host innate-immune reactive species. We have previously analyzed the bacterial response to nitrosative stress by S-nitrosoglutathione (GSNO), a physiological NO√ donor with diminished levels in the CF lung. GSNO substantially increased bacterial nitrosative and oxidative defenses and so we hypothesized a similar increase in alginate production would occur. However, in mucoid P. aeruginosa, there was decreased expression of the majority of alginate synthetic genes. This microarray data was confirmed both by RT-PCR and at the functional level by direct measurements of alginate production. Our data suggest that the lowered levels of innate-immune nitrosative mediators (such as GSNO) in the CF lung exacerbate the effects of mucoid P. aeruginosa, by failing to suppress alginate biosynthesis.     

Vascular oxidant stress: Molecular mechanisms and pathophysiological implications

The term oxidative stress refers to a situation in which cells are exposed to excessive levels of either molecular oxygen or chemical derivatives of oxygen (ie, reactive oxygen species). Three enzyme systems produce reactive oxygen species in the vascular wall: NADH/NADPH oxidase, xanthine oxidoreductase, and endothelial nitric oxide synthase. Among vascular reactive oxygen species superoxide anion plays a critical role in vascular biology because it is the source for many other reactive oxygen species and various vascular cell functions. It is currently thought that increases in oxidant stress, namely excessive production of superoxide anion, are involved in the pathophysiology of endothelial dysfunction that accompanies a number of cardiovascular risk factors including hypercholesterolemia, hypertension and cigarette smoking. On the other hand, vascular oxidant stress plays a pivotal role in the evolution of clinical conditions such as atherosclerosis, diabetes and heart failure.     

Nitrate tolerance does not increase production of peroxynitrite in the heart

Clinical studies have suggested that long-term nitrate treatment does not improve and may even worsen cardiovascular mortality, and the possible role of nitrate tolerance has been suspected. Nitrate tolerance has been recently shown to increase vascular superoxide and peroxynitrite production leading to vascular dysfunction. Nevertheless, nitrates exert direct cardiac effects independent from their vascular actions. Therefore, we investigated whether in vivo nitroglycerin treatment leading to vascular nitrate tolerance increases cardiac formation of nitric oxide (NO), reactive oxygen species, and peroxynitrite, thereby leading to cardiac dysfunction. Nitrate tolerance increased bioavailability of NO in the heart without increasing formation of reactive oxygen species. Despite elevated myocardial NO, neither cardiac markers of peroxynitrite formation nor cardiac mechanical function were affected by nitroglycerin treatment. However, serum free nitrotyrosine, a marker for systemic peroxynitrite formation, was significantly elevated in nitroglycerin-treated animals. This is the first demonstration that, although the systemic effects of nitroglycerin may be deleterious due to enhancement of extracardiac peroxynitrite formation, nitroglycerin does not result in oxidative damage in the heart.     

Nitrosative stress inhibits production of the virulence factor alginate in mucoid Pseudomonas aeruginosa

Alginate is a critical virulence factor contributing to the poor clinical prognosis associated with the conversion of Pseudomonas aeruginosa to mucoid phenotypes in cystic fibrosis (CF). An important mechanism of action is its ability to scavenge host innate-immune reactive species. We have previously analyzed the bacterial response to nitrosative stress by S-nitrosoglutathione (GSNO), a physiological NO√ donor with diminished levels in the CF lung. GSNO substantially increased bacterial nitrosative and oxidative defenses and so we hypothesized a similar increase in alginate production would occur. However, in mucoid P. aeruginosa, there was decreased expression of the majority of alginate synthetic genes. This microarray data was confirmed both by RT-PCR and at the functional level by direct measurements of alginate production. Our data suggest that the lowered levels of innate-immune nitrosative mediators (such as GSNO) in the CF lung exacerbate the effects of mucoid P. aeruginosa, by failing to suppress alginate biosynthesis.     

Emerging role of myeloperoxidase and oxidant stress markers in cardiovascular risk assessment

PURPOSE OF REVIEW: Myeloperoxidase, an abundant leukocyte protein that generates reactive oxidant species, is present and catalytically active within atherosclerotic lesions. Numerous lines of evidence suggest mechanistic links between myeloperoxidase, inflammation and both acute and chronic manifestations of cardiovascular disease. RECENT FINDINGS: Myeloperoxidase generates reactive oxidant species as part of its function in innate host defense mechanisms. The reactive species formed, however, may also damage normal tissues, contributing to inflammatory injury. Recent studies suggest that MPO-generated oxidants participate in multiple processes relevant to cardiovascular disease development and outcomes, including induction of foam cell formation, endothelial dysfunction, development of vulnerable plaque, and ventricular remodeling following acute myocardial infarction. Of note, measurements of myeloperoxidase mass and activity may be useful in cardiac risk stratification, both for chronic disease assessment, as well as in identification of patients at risk in the acute setting. SUMMARY: The inflammatory protein myeloperoxidase is present, active and mechanistically poised to participate in the initiation and progression of cardiovascular disease. The many links between myeloperoxidase, oxidation and cardiovascular disease suggest this leukocyte protein may have clinical utility in risk stratification for cardiovascular disease status and outcomes.     

Ankyrin-B Protein in Heart Failure: IDENTIFICATION OF A NEW COMPONENT OF METAZOAN CARDIOPROTECTION

Ankyrins (ankyrin-R, -B, and -G) are adapter proteins linked with defects in metazoan physiology. Ankyrin-B (encoded by ANK2) loss-of-function mutations are directly associated with human cardiovascular phenotypes including sinus node disease, atrial fibrillation, ventricular tachycardia, and sudden cardiac death. Despite the link between ankyrin-B dysfunction and monogenic disease, there are no data linking ankyrin-B regulation with common forms of human heart failure. Here, we report that ankyrin-B levels are altered in both ischemic and non-ischemic human heart failure. Mechanistically, we demonstrate that cardiac ankyrin-B levels are tightly regulated downstream of reactive oxygen species, intracellular calcium, and the calcium-dependent protease calpain, all hallmarks of human myocardial injury and heart failure. Surprisingly, β(II)-spectrin, previously thought to mediate ankyrin-dependent modulation in the nervous system and heart, is not coordinately regulated with ankyrin-B or its downstream partners. Finally, our data implicate ankyrin-B expression as required for vertebrate myocardial protection as hearts deficient in ankyrin-B show increased cardiac damage and impaired function relative to wild-type mouse hearts following ischemia reperfusion. In summary, our findings provide the data of ankyrin-B regulation in human heart failure, provide insight into candidate pathways for ankyrin-B regulation in acquired human cardiovascular disease, and surprisingly, implicate ankyrin-B as a molecular component for cardioprotection following ischemia.     

NADPH oxidases are in part responsible for increased cardiovascular superoxide production during aging

The aim of our study was to examine in rats, age-related differences in myocardial ischemic recovery and to determine the possible relationship with modification of cardiac and vascular oxidative stress. Isolated perfused hearts from young (2 months), adult (6 months), and old (21 months) Wistar rats were subjected to a ischemia–reperfusion sequence. Vascular histomorphological analyses were performed and NADPH oxidase was studied. The expression of angiotensin AT₁ receptors was evaluated using immunostaining. During the preischemic period, but also after ischemia, an aged-related decrease in myocardial functional parameters was observed, and was associated with an increased release of reactive oxygen species. In aortas, the activity and expression of NADPH oxidase increased with age according to the ESR, fluorescence microscopy, and immunohistochemistry; the NADPH oxidase involved was localized in endothelial cells. We found an age-related increase in the expression of endothelial angiotensin AT₁. Our study suggests that myocardial function and adaptation to ischemia–reperfusion declined during aging and are related to a higher level of oxidative stress. Endothelial NADPH oxidase is a major contributor to age-related cardiovascular deterioration. One of the regulators of vascular NADPH oxidase activity, the renin–angiotensin system, may be involved in the modulation of vascular superoxide production during the aging process.     

H(2)O(2)-induced O(2) production by a non-phagocytic NAD(P)H oxidase causes oxidant injury

Non-phagocytic NAD(P)H oxidases have been implicated as major sources of reactive oxygen species in blood vessels. These oxidases can be activated by cytokines, thereby generating O(2), which is subsequently converted to H(2)O(2) and other oxidant species. The oxidants, in turn, act as important second messengers in cell signaling cascades. We hypothesized that reactive oxygen species, themselves, can activate the non-phagocytic NAD(P)H oxidases in vascular cells to induce oxidant production and, consequently, cellular injury. The current report demonstrates that exogenous exposure of non-phagocytic cell types of vascular origin (smooth muscle cells and fibroblasts) to H(2)O(2) activates these cell types to produce O(2) via an NAD(P)H oxidase. The ensuing endogenous production of O(2) contributes significantly to vascular cell injury following exposure to H(2)O(2). These results suggest the existence of a feed-forward mechanism, whereby reactive oxygen species such as H(2)O(2) can activate NAD(P)H oxidases in non-phagocytic cells to produce additional oxidant species, thereby amplifying the vascular injury process. Moreover, these findings implicate the non-phagocytic NAD(P)H oxidase as a novel therapeutic target for the amelioration of the biological effects of chronic oxidant stress.     

NO Synthase and NO-Dependent Signal Pathways in Brain Aging and Neurodegenerative Disorders: The Role of Oxidant/Antioxidant Balance

Nitric oxide and other reactive nitrogen species appear to play several crucial roles in the brain. These include physiological processes such as neuromodulation, neurotransmission and synaptic plasticity, and pathological processes such as neurodegeneration and neuroinflammation. There is increasing evidence that glial cells in the central nervous system can produce nitric oxide in vivo in response to stimulation by cytokines and that this production is mediated by the inducible isoform of nitric oxide synthase. Although the etiology and pathogenesis of the major neurodegenerative and neuroinflammatory disorders (Alzheimer's disease, amyothrophic lateral sclerosis, Parkinson's disease, Huntington's disease and multiple sclerosis) are unknown, numerous recent studies strongly suggest that reactive nitrogen species play an important role. Furthermore, these species are probably involved in brain damage following ischemia and reperfusion, Down's syndrome and mitochondrial encephalopathies. Recent evidence also indicates the importance of cytoprotective proteins such as heat shock proteins (HSPs) which appear to be critically involved in protection from nitrosative and oxidative stress. In this review, evidence for the involvement of nitrosative stress in the pathogenesis of the major neurodegenerative/ neuroinflammatory diseases and the mechanisms operating in brain as a response to imbalance in the oxidant/antioxidant status are discussed.     

Urotensin II in the cardiovascular system

Urotensin II is a peptide present, together with its receptor, in the central nervous system and many peripheral tissues (including heart, blood vessels, kidneys and endocrine organs) of many species. The bioactive, mature form contains a cyclic heptapeptide perfectly preserved across species spanning 550 million years of evolution Its biological activity has been explored in cultured cells, in isolated vessels from several species, in the isolated perfused heart and in intact animals and man. Initial demonstration of potent vasoconstriction and cardiac depression by the human isoform in non-human primates has been followed by a series of reports indicating potent but highly variable and generally modest vascular responses dependent on species and vascular region. In man short term cardiovascular responses to administered urotensin II are small or absent. The place of urotensin II in the chronic trophic responses to cardiac and vascular injury and its possible roles as a neurotransmitter and/or regulator of renal and endocrine function remain largely unexplored.     

Chronic cigarette smoking causes hypertension, increased oxidative stress, impaired NO bioavailability, endothelial dysfunction, and cardiac remodeling in mice

Cigarette smoking is a major independent risk factor for cardiovascular disease. While the association between chronic smoking and cardiovascular disease is well established, the underlying mechanisms are incompletely understood, partly due to the lack of adequate in vivo animal models. Here, we report a mouse model of chronic smoking-induced cardiovascular pathology. Male C57BL/6J mice were exposed to whole body mainstream cigarette smoke (CS) using a SCIREQ "InExpose" smoking system (48 min/day, 5 days/wk) for 16 or 32 wk. Age-matched, air-exposed mice served as nonsmoking controls. Blood pressure was measured, and cardiac MRI was performed. In vitro vascular ring and isolated heart experiments were performed to measure vascular reactivity and cardiac function. Blood from control and smoking mice was studied for the nitric oxide (NO) decay rate and reactive oxygen species (ROS) generation. With 32 wk of CS exposure, mice had significantly less body weight gain and markedly higher blood pressure. At 32 wk of CS exposure, ACh-induced vasorelaxation was significantly shifted to the right and downward, left ventricular mass was significantly larger along with an increased heart-to-body weight ratio, in vitro cardiac function tended to be impaired with high afterload, white blood cells had significantly higher ROS generation, and the blood NO decay rate was significantly faster. Thus, smoking led to blunted weight gain, hypertension, endothelial dysfunction, leukocyte activation with ROS generation, decreased NO bioavailability, and mild cardiac hypertrophy in mice that were not otherwise predisposed to disease. This mouse model is a useful tool to enable further elucidation of the molecular and cellular mechanisms of smoking-induced cardiovascular diseases.     

Single exposure gamma-irradiation amplifies xanthine oxidase activity and induces endothelial dysfunction in rat aorta

Irradiation of the heart and vasculature can cause a spectrum of cardiovascular complications, including increased risk of myocardial infarction or coronary heart disease. Although irradiation is implicated in oxidant stress and chronic inflammation, the underlying molecular mechanisms have not been elucidated. We tested the hypothesis that irradiation-initiated upregulation of xanthine oxidase (XO), a primary source of cardiovascular reactive oxygen species, contributes to endothelial dysfunction and increased vascular stiffness. Twenty-two, 3-month-old Sprague–Dawley male rats were gamma-irradiated at the following doses: 0, 50, 160, and 500 cGy. Rats exposed to 500 cGy showed a significant increase in endothelial XO expression and a twofold increase in XO activity, compared to the 0 cGy controls. Endothelial function was investigated ex vivo through vascular tension dose–responses to the endothelial dependent vasodilator, acetylcholine. Endothelial-dependent relaxation in aorta of the 500 cGy exposed rats was significantly attenuated from the control group. Remarkably, specific inhibition of XO with oxypurinol restored the relaxation response to that of the control. Furthermore, these ex vivo results are reflected in vivo through alterations in vascular stiffness, as measured by pulse wave velocity (PWV). As early as 1-day post-exposure, rats exhibited a significant increase in PWV from pre-exposure. The PWV of irradiated rats (50, 160, and 500 cGy) were greater than those of 0 cGy control rats at 1 day, 1 and 2 weeks. The sham and irradiated rats possessed equivalent pre-exposure PWV, with sham showing no change over 2 weeks. Thus, these findings suggest that early upregulation of XO contributes to oxidative stress and endothelial nitro-redox imbalance with resultant endothelial dysfunction and altered vascular mechanics. Furthermore, these data identify XO as a potential molecular target for attenuating irradiation-induced cardiovascular injury.     

Endothelial mitochondria--a novel target for pharmacology of endothelial dysfunction

Vascular endothelium the inside layer of the cardiovascular system is presently looked upon as an important paracrine, autocrine and endocrine organ that determines the health of the cardiovascular system. In fact, healthy endothelium is essential for homeostasis of cardiovascular system, while endothelial dyfunction leads to cardiovascular diseases including atherosclerosis, diabetes and heart failure. Endothelial dysfunction is tightly linked to the overproduction of reactive oxygen species, development of oxidant stress and inflammatory response of endothelium. Mitochondria of the vascular endothelium seem to be an important player in these processes. In contrast to numerous cell types, synthesis of ATP in endothelium occurs in major part via a glycolytic pathway and endothelium seem to be relatively independent of the mitochondrial pathway of energy supply. However, as evident from recent studies, mitochondrial pathways of free radicals production tighly linked to mitochondrial and cytosol changes in the ion homeostasis play an important role in the regulation of endothelial inflammatory response, in the development of oxidative stress and apoptosis of vascular endothelium. Therefore, endothelial mitochondria appears critical in the regulation of endothelial functions and represent a novel target in pharmacology of endothelial dysfunction in cardiovascular diseases.     

Thyroid hormone and myocardial ischaemia

Thyroid hormone has various effects on the cardiovascular system and its effects on cardiac contractility, heart rhythm and vascular function has long been recognized. However, new evidence is emerged on the importance of thyroid hormone in the response of the myocardium to ischaemic stress and cardiac remodelling following myocardial infarction. Based on this new information, this review highlights the role of thyroid hormone in myocardial ischaemia and cardiac remodelling, the possible underlying mechanisms and the potential therapeutic implications. Thyroid hormone or analogs may prove new therapeutic agents for treating ischaemic heart disease.     

Oxidative and nitrosative stress in the maintenance of myocardial function

Reactive oxygen species (ROS) are generated by several different cellular sources, and their accumulation within the myocardium is widely considered to cause harmful oxidative stress. On the other hand, their role as second messengers has gradually emerged. The equilibrium of the nitroso/redox balance between reactive nitrogen species and ROS is crucial for the health of cardiomyocytes. This review provides a comprehensive overview of sources of oxidative stress in cardiac myocytes and describes the role of the nitroso/redox balance in cardiac pathophysiology. Although the exact mechanism of ROS production by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox's) is not completely understood, Nox2 and Nox4 have particularly important roles within the myocardium. Increasing evidence suggests that Nox2 produces superoxide and Nox4 generates only hydrogen peroxide. We also discuss the key role of nitric oxide synthases (NOSs) in the maintenance of the nitroso/redox balance: uncoupled endothelial NOS has been suggested to shift from nitric oxide to ROS production, contributing to increased oxidative stress within the myocardium. Furthermore, we highlight the importance of sequentially targeting and/or regulating the specific sources of oxidative and nitrosative stress to prevent and/or reverse myocardial dysfunction. Inhibition of NADPH oxidase-dependent ROS is considered to be a potential strategy for treatment of cardiomyopathy. Neither in vivo nor clinical data are available for NADPH oxidase inhibitors. Specifically targeting the mitochondria with the antioxidant MitoQ would be a very promising translation approach, because it could prevent mitochondrial permeability transition pore opening when ROS are produced during heart reperfusion. Enhancing NO signaling could also be a promising therapeutic approach against myocardial dysfunction.     

Thrombospondin-1 and CD47 regulation of cardiac,pulmonary and vascular responses in health and disease

Cardiovascular homeostasis and health is maintained through the balanced interactions of cardiac generated blood flow and cross-talk between the cellular components that comprise blood vessels. Central to this cross-talk is endothelial generated nitric oxide (NO) that stimulates relaxation of the contractile vascular smooth muscle (VSMC) layer of blood vessels. In cardiovascular disease this balanced interaction is disrupted and NO signaling is lost. Work over the last several years indicates that regulation of NO is much more complex than previously believed. It is now apparent that the secreted protein thrombospondin-1 (TSP1), that is upregulated in cardiovascular disease and animal models of the same, on activating cell surface receptor CD47, redundantly inhibits NO production and NO signaling. This inhibitory event has implications for baseline and disease-related responses mediated by NO. Further work has identified that TSP1-CD47 signaling stimulates enzymatic reactive oxygen species (ROS) production to further limit blood flow and promote vascular disease. Herein consideration is given to the most recent discoveries in this regard which identify the TSP1-CD47 axis as a major proximate governor of cardiovascular health.     

Cardiac and vascular phenotypes in the apolipoprotein E-deficient mouse

Cardiovascular death is frequently associated with atherosclerosis, a chronic multifactorial disease and a leading cause of death worldwide. Genetically engineered mouse models have proven useful for the study of the mechanisms underlying cardiovascular diseases. The apolipoprotein E-deficient mouse has been the most widely used animal model of atherosclerosis because it rapidly develops severe hypercholesterolemia and spontaneous atherosclerotic lesions similar to those observed in humans. In this review, we provide an overview of the cardiac and vascular phenotypes and discuss the interplay among nitric oxide, reactive oxygen species, aging and diet in the impairment of cardiovascular function in this mouse model.     

Effect of phenolic acids of microbial origin on production of reactive oxygen species in mitochondria and neutrophils

Cardiovascular death is frequently associated with atherosclerosis, a chronic multifactorial disease and a leading cause of death worldwide. Genetically engineered mouse models have proven useful for the study of the mechanisms underlying cardiovascular diseases. The apolipoprotein E-deficient mouse has been the most widely used animal model of atherosclerosis because it rapidly develops severe hypercholesterolemia and spontaneous atherosclerotic lesions similar to those observed in humans. In this review, we provide an overview of the cardiac and vascular phenotypes and discuss the interplay among nitric oxide, reactive oxygen species, aging and diet in the impairment of cardiovascular function in this mouse model.     

Reactive oxygen species and the Antarctic macroalgal wound response

Reactive oxygen species (ROS) are commonly produced by algal, vascular plant, and animal cells involved in the innate immune response as cellular signals promoting defense and healing and/or as a direct defense against invading pathogens. The production of reactive species in macroalgae upon injury, however, is largely uncharacterized. In this study, we surveyed 13 species of macroalgae from the Western Antarctic Peninsula and show that the release of strong oxidants is common after macroalgal wounding. Most species released strong oxidants within 1 min of wounding and/or showed cellular accumulation of strong oxidants over an hour post‐wounding. Exogenous catalase was used to show that hydrogen peroxide was a component of immediate oxidant release in one of five species, but was not responsible for the entire oxidative wound response as is common in vascular plants. The other component(s) of the oxidant cocktail released upon wounding are unknown. We were unable to detect protein nitration in extracts of four oxidant‐producing species flash frozen 30 s after wounding, but a role for reactive nitrogen species such as peroxynitrite cannot be completely ruled out. Two species showed evidence for the production of a catalase‐activated oxidant, a mechanism previously known only from the laboratory and from the synthetic drug isoniazid used to kill the human pathogen Mycobacterium tuberculosis. The rhodophyte Palmaria decipiens, which released strong oxidants after wounding, also produced strong oxidants upon grazing by a sympatric amphipod, suggesting that oxidants are involved in the response to grazing.