Clinical aspects of reactive oxygen and nitrogen species

Endothelial dysfunction in the setting of cardiovascular risk factors, such as hypercholesterolaemia, hypertension, diabetes mellitus and chronic smoking, as well as in the setting of heart failure, has been shown to be at least partly dependent on the production of reactive oxygen species in endothelial and/or smooth muscle cells and the adventitia, and the subsequent decrease in vascular bioavailability of NO. Superoxide-producing enzymes involved in increased oxidative stress within vascular tissue include NAD(P)H-oxidase, xanthine oxidase and endothelial nitric oxide synthase in an uncoupled state. Recent studies indicate that endothelial dysfunction of peripheral and coronary resistance and conductance vessels represents a strong and independent risk factor for future cardiovascular events. Ways to reduce endothelial dysfunction include risk-factor modification and treatment with substances that have been shown to reduce oxidative stress and, simultaneously, to stimulate endothelial NO production, such as inhibitors of angiotensin-converting enzyme or the statins. In contrast, in conditions where increased production of reactive oxygen species, such as superoxide, in vascular tissue is established, treatment with NO, e.g. via administration of nitroglycerin, results in a rapid development of endothelial dysfunction, which may worsen the prognosis in patients with established coronary artery disease.     

Expression of NOX-I, gp91phox, p47phox and P67phox in the aorta segments above and below coarctation

Aorta coarctation results in hypertension (HTN) in the arterial tree proximal to stenosis and, as such, provides an ideal model to discern the effects of different levels of blood pressure on the vascular tissue in the same animal. Compelling evidence has emerged supporting the role of oxidative stress as a cause of HTN. However, whether or not HTN (independent of the circulating humoral factors) can cause oxidative stress is less certain. NAD(P)H oxidase isoforms are the main source of reactive oxygen species (ROS) in the vascular tissues. We therefore compared the expressions of NOX-I, gp91phox and the regulatory subunits of the enzyme in the aorta segments residing above and below coarctation in rats with abdominal aorta banding. Rats were studied 4 weeks after aorta banding above the renal arteries or sham operation. Subunits of NAD(P)H oxidase and its NOX-I isoform as well as endothelial NO synthase (eNOS) and nitrotyrosine (footprint of NO oxidation by superoxide) were measured in the aorta segments above and below coarctation. The gp91phox, p47phox, and p67phox subunits of NAD(P)H oxidase, NOX-I isoform, eNOS and nitrotyrosine were markedly increased in the aorta segment above coarctation (hypertensive zone), but were virtually unchanged in the segment below coarctation. Since, excepting blood pressure, all other conditions were constant, the upregulation of NAD(P)H oxidase isoforms and the increased NO oxidation in the aorta segment above, but not below, coarctation prove that HTN, per se, independent of circulating mediators can cause oxidative/nitrosative stress in the arterial wall. These observations suggest that HTN control may represent a specific form of antioxidant therapy for hypertensive disorders.     

Reactive oxygen species in vascular biology: implications in hypertension

Reactive oxygen species (ROS), including superoxide (·O₂^–), hydrogen peroxide (H₂O₂), and hydroxyl anion (OH-), and reactive nitrogen species, such as nitric oxide (NO) and peroxynitrite (ONOO^–), are biologically important O₂ derivatives that are increasingly recognized to be important in vascular biology through their oxidation/reduction (redox) potential. All vascular cell types (endothelial cells, vascular smooth muscle cells, and adventitial fibroblasts) produce ROS, primarily via cell membrane-associated NAD(P)H oxidase. Reactive oxygen species regulate vascular function by modulating cell growth, apoptosis/anoikis, migration, inflammation, secretion, and extracellular matrix protein production. An imbalance in redox state where pro-oxidants overwhelm anti-oxidant capacity results in oxidative stress. Oxidative stress and associated oxidative damage are mediators of vascular injury and inflammation in many cardiovascular diseases, including hypertension, hyperlipidemia, and diabetes. Increased generation of ROS has been demonstrated in experimental and human hypertension. Anti-oxidants and agents that interrupt NAD(P)H oxidase-driven ·O₂^– production regress vascular remodeling, improve endothelial function, reduce inflammation, and decrease blood pressure in hypertensive models. This experimental evidence has evoked considerable interest because of the possibilities that therapies targeted against reactive oxygen intermediates, by decreasing generation of ROS and/or by increasing availability of antioxidants, may be useful in minimizing vascular injury and hypertensive end organ damage. The present chapter focuses on the importance of ROS in vascular biology and discusses the role of oxidative stress in vascular damage in hypertension.     

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.     

Angiotensin II-induced reduction in exercise capacity is associated with increased oxidative stress in skeletal muscle

Angiotensin II (ANG II)-induced oxidative stress has been known to be involved in the pathogenesis of cardiovascular diseases. We have reported that the oxidative stress in skeletal muscle can limit exercise capacity in mice (16). We thus hypothesized that ANG II could impair the skeletal muscle energy metabolism and limit exercise capacity via enhancing oxidative stress. ANG II (50 ng·kg(-1)·min(-1)) or vehicle was infused into male C57BL/6J mice for 7 days via subcutaneously implanted osmotic minipumps. ANG II did not alter body weight, skeletal muscle weight, blood pressure, cardiac structure, or function. Mice were treadmill tested, and expired gases were analyzed. The work to exhaustion (vertical distance × body weight) and peak oxygen uptake were significantly decreased in ANG II compared with vehicle. In mitochondria isolated from skeletal muscle, ADP-dependent respiration was comparable between ANG II and vehicle, but ADP-independent respiration was significantly increased in ANG II. Furthermore, complex I and III activities were decreased in ANG II. NAD(P)H oxidase activity and superoxide production by lucigenin chemiluminescence were significantly increased in skeletal muscle from ANG II mice. Treatment of ANG II mice with apocynin (10 mmol/l in drinking water), an inhibitor of NAD(P)H oxidase activation, completely inhibited NAD(P)H oxidase activity and improved exercise capacity, mitochondrial respiration, and complex activities in skeletal muscle. ANG II-induced oxidative stress can impair mitochondrial respiration in skeletal muscle and limit exercise capacity.     

Upregulation of the vascular NAD(P)H-oxidase isoforms Nox1 and Nox4 by the renin-angiotensin system in vitro and in vivo.

In different cardiovascular disease states, oxidative stress decreases the bioavailability of endothelial NO, resulting in endothelial dysfunction. An important molecular source of reactive oxygen species is the enzyme family of NAD(P)H oxidases (Nox). Here we provide evidence that the vascular Nox isoforms Nox1 and Nox4 appear to be involved in vascular oxidative stress in response to risk factors like angiotensin II (Ang II) in vitro as well as in vivo. Nox mRNA and protein levels were quantified by real-time RT-PCR and Western blotting, respectively. Nox1 and Nox4 were expressed in the vascular smooth muscle cell (VSMC) line A7r5 and aortas and kidneys of rats. Upon exposure of A7r5 cells to Ang II (1 microM, 4 h), Nox1 and Nox4 mRNA levels were increased 6-fold and 4-fold, respectively. Neither the vasoconstrictor endothelin 1 (up to 500 nM, 1-24 h) nor lipopolysaccharide (up to 100 ng/ml, 1-24 h) had any effect on Nox1 and Nox4 expression in these cells. Consistent with these observations made in vitro, aortas and kidneys of transgenic hypertensive rats overexpressing the Ren2 gene [TGR(mRen2)27] had significantly higher amounts of Nox1 and Nox4 mRNA and of Nox4 protein compared to tissues from normotensive wild-type animals. In conclusion, Nox4 and Nox1 are upregulated by the renin-angiotensin system. Increased superoxide production by upregulated vascular Nox isoforms may diminish the effectiveness of NO and thus contribute to the development of vascular diseases. Nox1 and Nox4 could be targeted therapeutically to reduce vascular reactive oxygen species production and thereby increase the bioavailability of NO.     

Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system

The renin-angiotensin system is a central component of the physiological and pathological responses of cardiovascular system. Its primary effector hormone, angiotensin II (ANG II), not only mediates immediate physiological effects of vasoconstriction and blood pressure regulation, but is also implicated in inflammation, endothelial dysfunction, atherosclerosis, hypertension, and congestive heart failure. The myriad effects of ANG II depend on time (acute vs. chronic) and on the cells/tissues upon which it acts. In addition to inducing G protein- and non-G protein-related signaling pathways, ANG II, via AT₁ receptors, carries out its functions via MAP kinases (ERK 1/2, JNK, p38MAPK), receptor tyrosine kinases [PDGF, EGFR, insulin receptor], and nonreceptor tyrosine kinases [Src, JAK/STAT, focal adhesion kinase (FAK)]. AT₁R-mediated NAD(P)H oxidase activation leads to generation of reactive oxygen species, widely implicated in vascular inflammation and fibrosis. ANG II also promotes the association of scaffolding proteins, such as paxillin, talin, and p130Cas, leading to focal adhesion and extracellular matrix formation. These signaling cascades lead to contraction, smooth muscle cell growth, hypertrophy, and cell migration, events that contribute to normal vascular function, and to disease progression. This review focuses on the structure and function of AT₁ receptors and the major signaling mechanisms by which angiotensin influences cardiovascular physiology and pathology. vascular smooth muscle; NAD(P)H oxidase; tyrosine and nontyrosine receptor kinases; endothelial dysfunction; vascular disease     

Increased expression of NAD(P)H oxidase in islets of animal models of Type 2 diabetes and its improvement by an AT1 receptor antagonist

This study was undertaken to reveal the role of NAD(P)H oxidase in increased oxidative stress in islets of Type 2 diabetes. Immunostaining analysis showed that staining intensities of NAD(P)H oxidase components, gp91phox and p22phox, significantly increased in islets of animal models of Type 2 diabetes, OLETF rats (60 weeks of age) and db/db mice (14 weeks of age), compared with age-matched controls, respectively, correlating with increased levels of oxidative stress marker, 8-hydroxy-deoxyguanosine or 4-hydroxy-2-nonenal modified protein. In db/db mice, oral administration of angiotensin II Type 1 receptor antagonist valsartan (5 mg/kg) for 4 weeks significantly attenuated the increased expression of gp91phox and p22phox together with inhibition of oxidative stress and partially restored decreased insulin contents in islets. Angiotensin II-related increased expression of NAD(P)H oxidase may play an important role in increased oxidative stress in islets of Type 2 diabetes. This mechanism may be a novel therapeutic target for preventing beta-cell damage.     

NAD(P)H oxidase inhibitor prevents blood pressure elevation and cardiovascular hypertrophy in aldosterone-infused rats

Increased bioavailability of reactive oxygen species (ROS) has been implicated in the pathogenesis of mineralocorticoid hypertension. To find out the source of ROS, we evaluated the role of NAD(P)H oxidase in blood pressure (BP) elevation, cardiovascular hypertrophy, and fibrosis in aldosterone-salt rats. Aldosterone infusion (0.75 microg/h) significantly increased BP, which is attenuated by apocynin (1.5 mmol/L). Cardiac hypertrophy developed by aldosterone infusion was also normalized with apocynin. Greater mRNA for p22phox and NAD(P)H oxidase activity (more than twofold) in aorta of aldosterone-infused rats was reduced in apocynin-treated rats. Aldosterone infusion increased marginally procollagen I and III expression in LV compared to controls and apocynin decreased procollagen. Masson's Trichrome stain showed increased cardiac perivascular fibrosis, which was reduced by apocynin. These results suggest that NAD(P)H oxidase plays an important role in cardiovascular damage associated with mineralocorticoid hypertension.     

Vascular superoxide and hydrogen peroxide production and oxidative stress resistance in two closely related rodent species with disparate longevity

Vascular aging is characterized by increased oxidative stress, impaired nitric oxide (NO) bioavailability and enhanced apoptotic cell death. The oxidative stress hypothesis of aging predicts that vascular cells of long-lived species exhibit lower production of reactive oxygen species (ROS) and/or superior resistance to oxidative stress. We tested this hypothesis using two taxonomically related rodents, the white-footed mouse (Peromyscus leucopus) and the house mouse (Mus musculus), that show a more than twofold difference in maximum lifespan potential (MLSP = 8 and 3.5 years, respectively). We compared interspecies differences in endothelial superoxide (O2-) and hydrogen peroxide (H2O2) production, NAD(P)H oxidase activity, mitochondrial ROS generation, expression of pro- and antioxidant enzymes, NO production, and resistance to oxidative stress-induced apoptosis. In aortas of P. leucopus, NAD(P)H oxidase expression and activity, endothelial and H2O2 production, and ROS generation by mitochondria were less than in mouse vessels. In P. leucopus, there was a more abundant expression of catalase, glutathione peroxidase 1 and hemeoxygenase-1, whereas expression of Cu/Zn-SOD and Mn-SOD was similar in both species. NO production and endothelial nitric oxide synthase expression was greater in P. leucopus. In mouse aortas, treatment with oxidized low-density lipoprotein (oxLDL) elicited substantial oxidative stress, endothelial dysfunction and endothelial apoptosis (assessed by TUNEL assay, DNA fragmentation and caspase 3 activity assays). According to our prediction, vessels of P. leucopus were more resistant to the proapoptotic effects of oxidative stressors (oxLDL and H2O2). Primary fibroblasts from P. leucopus also exhibited less H2O2-induced DNA damage (comet assay) than mouse cells. Thus, increased lifespan potential in P. leucopus is associated with a decreased cellular ROS generation and increased oxidative stress resistance, which accords with the prediction of the oxidative stress hypothesis of aging.     

Regulation of NAD(P)H oxidases by AMPK in cardiovascular systems

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are ubiquitously produced in cardiovascular systems. Under physiological conditions, ROS/RNS function as signaling molecules that are essential in maintaining cardiovascular function. Aberrant concentrations of ROS/RNS have been demonstrated in cardiovascular diseases owing to increased production or decreased scavenging, which have been considered common pathways for the initiation and progression of cardiovascular diseases such as atherosclerosis, hypertension, (re)stenosis, and congestive heart failure. NAD(P)H oxidases are primary sources of ROS and can be induced or activated by all known cardiovascular risk factors. Stresses, hormones, vasoactive agents, and cytokines via different signaling cascades control the expression and activity of these enzymes and of their regulatory subunits. But the molecular mechanisms by which NAD(P)H oxidase is regulated in cardiovascular systems remain poorly characterized. Investigations by us and others suggest that adenosine monophosphate-activated protein kinase (AMPK), as an energy sensor and modulator, is highly sensitive to ROS/RNS. We have also obtained convincing evidence that AMPK is a physiological suppressor of NAD(P)H oxidase in multiple cardiovascular cell systems. In this review, we summarize our current understanding of how AMPK functions as a physiological repressor of NAD(P)H oxidase.     

Integrin-linked kinase is a central mediator in angiotensin II type 1- and chemokine receptor CXCR4 signaling in myocardial hypertrophy

Inflammation and pro-hypertrophic signaling are important for development and progression of myocardial hypertrophy (LVH) and chronic heart failure (CHF). Here we investigated the relevance of integrin-linked kinase (ILK) for chemokine receptor CXCR4- and angiotensin II type 1-triggered signaling and its regulation and role in cardiac remodeling.Using ELISA, real-time-PCR, and Western blotting, the present study demonstrates that SDF-1 and its receptor CXCR4 are up-regulated in plasma and left ventricles, respectively, in mouse models of cardiac hypertrophy (transaortic constriction, transgenic cardiac-specific overexpression of rac1) and in human CHF in association with increased cardiac ILK-expression. In isolated cardiomyocytes, ILK is activated by CXCR4-ligation and necessary for SDF-1-triggered activation of rac1, NAD(P)H oxidase, and release of reactive oxygen species. Importantly, the pro-hypertrophic peptide angiotensin II induces ILK-activation dependent on rac1 in cardiomyocytes, where ILK is necessary for angiotensin II-mediated stimulation of hypertrophy genes and protein synthesis.We conclude that in both SDF-1- and angiotensin II-triggered signaling, ILK is a central mediator of rac1-induced oxidative stress and myocardial hypertrophy.     

Short-term exposure of high glucose concentration induces generation of reactive oxygen species in endothelial cells: implication for the oxidative stress associated with postprandial hyperglycemia

Recent studies demonstrating a close relationship between postprandial hyperglycemia and the incidence of atherosclerotic cardiovascular disease prompted us to investigate the generation and source of reactive oxygen species (ROS) in endothelial cells stimulated by short-term exposure to a high glucose concentration. In addition, we investigated the effect of insulin on ROS production induced by high glucose concentration. Cultured bovine aortic endothelial cells demonstrated a significant increase in intracellular ROS generation after a 3-h exposure to 25 mM glucose (131.4% versus 5 mM glucose). This increased generation of ROS was suppressed by an inhibitor of NAD(P)H oxidase. Intracellular ROS production in cells exposed to 3 h of high glucose concentration was increased significantly by the presence of a physiological concentration of insulin. However, after a 1-h exposure to high glucose levels, ROS generation in cells incubated with insulin was only about 80% of that measured in cells incubated without insulin. The generation of intracellular nitric oxide (NO) resulting from an acute insulin effect may account for this difference. In conclusion, acute hyperglycemia itself may possibly cause endothelial oxidative stress in patients with postprandial hyperglycemia. Endothelial oxidative stress may be determined by the interaction between NO and superoxide generation.     

Light-induced decrease in DCF fluorescence of wheat leaves in the presence of salicyl hydroxamate

5-(and-6)-Carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), a permeative indicator of oxidative stress, was loaded into dissected leaves of wheat in order to monitor the temporal development of reactive oxygen species. DCF fluorescence was found to be constant under dark conditions. Upon loading the leaves with salicyl hydroxamate, a blocker of the alternative oxidase, DCF fluorescence linearly increased in the dark. This indicates a function of alternative oxidase in preventing reactive oxygen radicals in the mitochondria. Upon illumination, the DCF signal decreased within 5 min. As illuminated chloroplasts would increase the load of reactive oxygen species, the observed decrease cannot be assigned to the production of reactive oxygen species in the chloroplasts. Three different putative mechanisms are considered which all assign an important role to light-induced delivery of NAD(P)H: (1) direct quenching of DCF fluorescence by light-generated NAD(P)H, (2) light-stimulated activation of scavenging enzymes, or (3) redirection of mitochondrial electron fluxes as caused by the delivery of excess redox equivalents (NADH) from the chloroplasts.     

Reactive oxygen species as mediators of angiogenesis signaling. Role of NAD(P)H oxidase

Angiogenesis, a process of new blood vessel growth, contributes to various pathophysiologies such as cancer, diabetic retinopathy and atherosclerosis. Accumulating evidence suggests that cardiovascular diseases are associated with increased oxidative stress in blood vessels. Reactive oxygen species (ROS) such as superoxide and H2O2 cause blood vessels to thicken, produce inflammation in the vessel wall, and thus are regarded as "risk factors" for vascular disease, whereas ROS also act as signaling molecules in many aspects of growth factor-mediated physiological responses. Recent reports suggest that ROS play an important role in angiogenesis; however, its underlying molecular mechanisms remain unknown. Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell (EC) proliferation and migration primarily through the receptor tyrosine kinase VEGF receptor2 (Flk1/KDR). VEGF binding initiates tyrosine phosphorylation of KDR, which results in activation of downstream signaling enzymes including ERK1/2, Akt and eNOS, which contribute to angiogenic-related responses in EC. Importantly, the major source of ROS in EC is a NAD(P)H oxidase and EC express all the components of phagocytic NAD(P)H oxidase including gp91phox, p22phox, p47phox, p67phox and the small G protein Rac1. We have recently demonstrated that ROS derived from NAD(P)H oxidase are critically important for VEGF signaling in vitro and angiogenesis in vivo. Furthermore, a peptide hormone, angiotensin II, a major stimulus for vascular NAD(P)H oxidase, also plays an important role in angiogenesis. Because EC migration and proliferation are primary features of the process of myocardial angiogenesis, we would like to focus on the recent progress that has been made in the emerging area of NAD(P)H oxidase-derived ROS-dependent signaling in ECs, and discuss the possible roles in angiogenesis. Understanding these mechanisms may provide insight into the components of NAD(P)H oxidase as potential therapeutic targets for treatment of angiogenesis-dependent diseases such as cancer and atherosclerosis and for promoting myocardial angiogenesis in ischemic heart diseases.     

Reactive oxygen species as mediators of angiotensin II signaling

Angiotensin II stimulates a plethora of signaling pathways leading to cell growth and contraction. Recent work has shown that reactive oxygen species are involved in transducing many of the effects of angiotensin II, and are in fact produced in response to agonist-receptor binding. Angiotensin II stimulates a NAD(P)H oxidase to produce superoxide and hydrogen peroxide, both of which may act on intracellular growth-related proteins and enzymes to mediate the final physiological response. Of particular importance is hydrogen peroxide, which mediates angiotensin II stimulation of such important intracellular signals as EGF-receptor transactivation, p38 mitogen activated protein kinase, and Akt. Future work will be directed towards identifying other important redox-sensitive signaling pathways and their relationship to the physiology and pathophysiology of the renin-angiotensin system.     

NAD(P)H oxidase-derived hydrogen peroxide mediates endothelial nitric oxide production in response to angiotensin II

Recently, it has been shown that the exogenous addition of hydrogen peroxide (H(2)O(2)) increases endothelial nitric oxide (NO(.)) production. The current study is designed to determine whether endogenous levels of H(2)O(2) are ever sufficient to stimulate NO(.) production in intact endothelial cells. NO(.) production was detected by a NO(.)-specific microelectrode or by an electron spin resonance spectroscopy using Fe(2+)-(DETC)(2) as a NO(.)-specific spin trap. The addition of H(2)O(2) to bovine aortic endothelial cells caused a potent and dose-dependent increase in NO(.) release. Incubation with angiotensin II (10(-7) mol) elevated intracellular H(2)O(2) levels, which were attenuated with PEG-catalase. Angiotensin II increased NO(.) production by 2-fold, and this was prevented by Losartan and by PEG-catalase, suggesting a critical role of AT1 receptor and H(2)O(2) in this response(.) In contrast, NO(.) production evoked by either bradykinin or calcium ionophore was unaffected by PEG-catalase. As in bovine aortic endothelial cells, angiotensin II doubled NO(.) production in aortic endothelial cells from C57BL/6 mice but had no effect on NO(.) production in endothelial cells from p47(phox-/-) mice. In contrast, stimulated NO(.) production to a similar extent in endothelial cells from wild-type and p47(phox-/-) mice. In summary, the present study provides direct evidence that endogenous H(2)O(2), derived from the NAD(P)H oxidase, mediates endothelial NO(.) production in response to angiotensin II. Under disease conditions associated with elevated levels of angiotensin II, this response may represent a compensatory mechanism. Because angiotensin II also stimulates O(2)() production from the NAD(P)H oxidase, the H(2)O(2) stimulation of NO(.) may facilitate peroxynitrite formation in response to this octapeptide.     

Dual role of plasma membrane electron transport systems in defense

Because oxidative stress is one of the main sources of severe cellular damage, cells have different defense weapons against reactive oxygen species. Ubiquitous plasma membrane redox systems play a role in defense against oxidative stress damage. On the other hand, a tightly controlled and localized production of reactive oxygen species by a plasma membrane NADPH oxidase can be used as a potent microbicidal weapon. This dual, prooxidant and antioxidant role of plasma membrane electron transport systems in defense is studied and discussed.