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It is not clear whether endothelial cell (EC) activation by the hormone angiotensin II (Ang II) modulates contraction of vascular smooth muscle ceils (VSMCs) in the vasculature and whether impairment of this regulation in vivo contributes to hypertension. Delineation of the actions of Ang II through the type 1 receptor (AT1R) on ECs in the blood vessels has been a challenging problem because of the predominance of the AT1R functions in VSMCs that lie underneath the endothelium. We have obviated this limitation by generating transgenic (TG) mice engineered to target expression of the constitutively active N111G mutant AT1R only in ECs. In these TG mice, the enhanced angiotensinergic signal in ECs without infusion of Ang II resulted in hypotension and bradycardia. The pressor response to acute infusion of Ang II was significantly reduced. Increased expression of endothelial nitric oxide synthase and production of hypotensive mediators, nitric oxide and cyclic guanosine monophosphate, cause these phenotypes. Hypotension and bradycardia observed in the TG mice could be rescued by treatment with an AT1R-selective antagonist. Our results imply that the Ang II action by means of EC-AT1R is antagonistic to vasoconstriction in general, and it may moderate the magnitude of functional response to Ang II in VSMCs. This control mechanism in vivo most likely is a determinant of altered hemodynamic regulation involved in endothelial dysfunction in hypertensive cardiovascular disease. angiotensin II receptor | endothelial nitric oxide synthase | hypertension | nitric oxide | angiotensin receptor blockers

Protein levels and polymorphisms of p[22.sup.phox] have been suggested to modulate vascular NAD(P)H oxidase activity and vascular production of reactive oxygen species (ROS). We sought to determine whether increasing p[22.sup.phox] expression would alter vascular ROS production and hemodynamics by targeting p[22.sup.phox] expression to smooth muscle in transgenic (Tg) mice. Aortas of [Tg.sup.p22smc] mice had increased p[22.sup.phox] and Nox1 protein levels and produced more superoxide and [H.sub.2][O.sub.2]. Surprisingly, endothelium-dependent relaxation and blood pressure in [Tg.sup.p22smc] mice were normal. Aortas of [Tg.sup.p22smc] mice produced twofold more nitric oxide (NO) at baseline and sevenfold more NO in response to calcium ionophore as detected by electron spin resonance. Western blot analysis revealed a twofold increase in endothelial NO synthase (eNOS) protein expression in [Tg.sup.p22smc] mice. Both eNOS expression and NO production were normalized by infusion of the glutathione peroxidase mimetic ebselen or by crossing [Tg.sup.p22smc] mice with mice overexpressing catalase. We have previously found that NO stimulates extracellular superoxide dismutase (ecSOD) expression in vascular smooth muscle. In keeping with this, aortic segments from [Tg.sup.p22smc] mice expressed twofold more ecSOD, and chronic treatment with the NOS inhibitor [N.sup.G]-nitro-L-arginine methyl ester normalized this, suggesting that NO regulates ecSOD protein expression in vivo. These data indicate that chronic oxidative stress caused by excessive [H.sub.2][O.sub.2] production evokes a compensatory response involving increased eNOS expression and NO production. NO in turn increases ecSOD protein expression and counterbalances increased ROS production leading to the maintenance of normal vascular function and hemodynamics. transgenic animals; oxidant stress; endothelium; nitric oxide

Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay. Am J Physiol Cell Physiol 287: C895-C902, 2004. First published August 11, 2004; 10.1152/ajpcell.00028.2004.--Recently, it was demonstrated that superoxide oxidizes dihydroethidium to a specific fluorescent product (oxyethidium) that differs from ethidium by the presence of an additional oxygen atom in its molecular structure (Zhao H, Kalivendi S, Zhang H, Joseph J, Nithipatikom K, Vasquez-Vivar J, and Kalyanaraman B. Free Radic Biol Med 34: 1359-1368, 2003). We have adapted this new HPLC-based assay to quantify this product as a tool to estimate intracellular superoxide in intact tissues. Ethidium and oxyethidium were separated using a C-18 column and quantified using fluorescence detection. Initial cell-free experiments with potassium superoxide and xanthine oxidase confirmed the formation of oxyethidium from dihydroethidium. The formation of oxyethidium was inhibited by superoxide dismutase but not catalase and did not occur upon the addition of [H.sub.2][O.sub.2], peroxynitrite, or hypochlorous acid. In bovine aortic endothelial cells (BAEC) and murine aortas, the redox cycling drug menadione increased the formation of oxyethidium from dihydroethidium ninefold (0.4 nmol/mg in control vs. 3.6 nmol/mg with 20 [micro]M menadione), and polyethylene glycolconjugated superoxide dismutase (PEG-SOD) significantly inhibited this effect. Treatment of BAEC with angiotensin II caused a twofold increase in oxyethidium formation, and this effect also was reduced by PEG-SOD (0.5 nmol/mg). In addition, in the aortas of mice with angiotensin II-induced hypertension and DOCA-salt hypertension, the formation of oxyethidium was increased in a manner corresponding to superoxide production estimated on the basis of cytochrome c reduction. Detection of oxyethidium using HPLC represents a new, convenient, quantitative method for the detection of superoxide in intact cells and tissues. oxyethidium: hypertension; menadione; angiotensin II; endothelium

The main purpose of this study was to investigate the acute local and systemic effects of low-load resistance exercise (30% 1RM) with partial vascular occlusion on exercise-induced free radical production and to compare these effects with other established training methods. Fifteen young and healthy males (25 ± 3 years) performed the following four sessions in a counterbalanced order on separate days: low-load resistance exercise (LI: 30% 1RM), low-load resistance exercise with blood flow restriction (LIBR: 30% 1RM), high-load resistance exercise (HI: 80% 1RM) and an additional session without exercise but blood flow restriction only (BR). Blood samples were obtained 15 min prior to and immediately after exercise sessions from the right index finger and first toe. To analyze concentrations of reactive oxygen species (ROS), electron paramagnetic resonance (EPR) spectroscopy was used. Additionally, mitochondrial ROS production was measured by adding inhibitors of electron transport chain complex III. There was an increased systemic ROS generation after the LIBR session from 0.837 ± 0.093 to 0.911 ± 0.099 µmol/l/min. However, no local or systemic time × condition interaction was detected for ROS production. Regarding mitochondrial ROS production, results were not different between the conditions. Although the low-load resistance exercise session with partial vascular occlusion elicited systemic increases of ROS production, no significant changes were seen on a local level. We assume that this ROS concentration might not be high enough to induce cellular damage but is rather involved in muscle remodulation. However, this needs to be confirmed by future research. [ABSTRACT FROM AUTHOR]

Recent interest has focused on maintenance of healthy levels of redox signalling and the related oxidants; these parameters are crucial for providing us with concrete nutritional targets that may help us to better understand and maintain “optimal health”. Following the above hypothesis, we performed a pilot double-blind, crossover, placebo-controlled, single dose study to measure the dose-dependent effects of a proprietary plant-based dietary supplement labelled here as S7 (SPECTRA7), related to how it affected the cellular metabolic index (CMI) in healthy human participants (n = 8). We demonstrated using the electron spin resonance/electron paramagnetic resonance spectrometer NOXYSCAN that the administration S7 resulted in statistically significant, long-term, dose-dependent inhibition of mitochondrial and cellular reactive oxygen species generation by as much as 9.2 or 17.7% as well as 12.0 or 14.8% inhibition in extracellular nicotinamide-dinucleotide-phosphate oxidase system-dependent generation of O2•−, and 9.5 or 44.5% inhibition of extracellular H2O2 formation. This was reflected with dose-dependent 13.4 or 17.6% inhibition of tumour necrosis factor alpha induced cellular inflammatory resistance and also 1.7 or 2.3-times increases of bioavailable NO concentration. In this pilot study, we demonstrated the ability of a natural supplement to affect cellular redox signalling, which is considered by many researchers as oxidative stress. The design and activity of this proprietary plant-based material, in combination with the newly developed “CMI” test, demonstrates the potential of using dietary supplements to modulate redox signalling. This opens the door to future research into the use of S7 for modulation of inflammatory markers, for sports endurance or recovery applications. [ABSTRACT FROM AUTHOR]

The research community is generally agreed that maintenance of healthy levels of free radicals and related oxidants are important for good health. However, utilization of the 'redox stress hypothesis' can provide us with concrete nutritional targets in order to better support and maintain 'optimal health.' Following this hypothesis we performed a crossover, double-blind, placebo-controlled, single-dose study on the effects of SPECTRA™, a dietary supplement, on oxidative stress markers (OSM) in human participants ( n = 22). The measurement of OSM (ex vivo intra- and extracellular formation of reactive oxygen species (ROS, O2−, H2O2, OH−) in whole blood, respiratory activity of blood cells, as well as mitochondrial-dependent ROS formation, and respiratory activity), was performed using EPR spectrometer nOxyscan, spin probe CMH, and oxygen label NOX-15.1, respectively. Furthermore, we investigated the ability of SPECTRA™ to modulate ex vivo cellular inflammatory responses induced by stimulation with exogenous TNF- α and also followed changes in bioavailable NO concentrations. In this clinical study, we demonstrated that administration of SPECTRA™ resulted in statistically significant long-term inhibition of mitochondrial and cellular ROS generation by as much as 17% as well as 3.5-times inhibition in extracellular NADPH system-dependent generation of O2−, and nearly complete inhibition of extracellular H2O2 formation. This was reflected in more than two times inhibition of ex vivo cellular inflammatory response and also increases in bioavailable NO concentration. For the first time, we have measured synergetic, biological effects of a natural supplement on changes in OSM and cellular metabolic activity. The unique design and activity of the plant-based natural supplement, in combination with the newly developed and extended Vitality test, demonstrates the potential of using dietary supplements to modulate OSM and also opens the door to future research into the use of natural supplements for supporting optimal health. [ABSTRACT FROM AUTHOR]

The brain contains a highly diversified complement of molecular species of a mitochondria-specific phospholipid, cardiolipin, which, because of its polyunsaturation, can readily undergo oxygenation. Using global lipidomics analysis in experimental traumatic brain injury (TBI), we found that TBI was accompanied by oxidative consumption of polyunsaturated cardiolipin and the accumulation of more than 150 new oxygenated molecular species of cardiolipin. RNAi-based manipulations of cardiolipin synthase and cardiolipin levels conferred resistance to mechanical stretch, an in vitro model of traumatic neuronal injury, in primary rat cortical neurons. By applying a brain-permeable mitochondria-targeted electron scavenger, we prevented cardiolipin oxidation in the brain, achieved a substantial reduction in neuronal death both in vitro and in vivo, and markedly reduced behavioral deficits and cortical lesion volume. We conclude that cardiolipin oxygenation generates neuronal death signals and that prevention of it by mitochondria-targeted small molecule inhibitors represents a new target for neuro-drug discovery. [ABSTRACT FROM AUTHOR]

Background: Fetal Growth Restriction is often associated with a feto-placental vascular dysfunction conceivably involving endothelial cells. Our study aimed to verify this pathogenic role for feto-placental endothelial cells and, coincidentally, demonstrate any abnormality in the nitric oxide system. Methods: Prenatal assessment of feto-placental vascular function was combined with measurement of nitric oxide (in the form of S-nitrosohemoglobin) and its nitrite byproduct, and of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine. Umbilical vein endothelial cells were also harvested to determine their gene profile. The study comprised term pregnancies with normal (n = 40) or small-for-gestational-age (n = 20) newborns, small-for-gestational-age preterm pregnancies (n = 15), and bi-chorial, bi-amniotic twin pregnancies with discordant fetal growth (n = 12). Results: Umbilical blood nitrite (p<0.001) and S-nitrosohemoglobin (p = 0.02) rose with fetal growth restriction while asymmetric dimethylarginine decreased (p = 0.003). Nitrite rise coincided with an abnormal Doppler profile from umbilical arteries. Fetal growth restriction umbilical vein endothelial cells produced more nitrite and also exhibited reciprocal changes in vasodilator (upwards) and vasoconstrictor (downwards) transcripts. Elevation in blood nitrite and S-nitrosohemoglobin persisted postnatally in the fetal growth restriction offspring. Conclusion: Fetal growth restriction is typified by increased nitric oxide production during pregnancy and after birth. This response is viewed as an adaptative event to sustain placental blood flow. However, its occurrence may modify the endothelial phenotype and may ultimately represent an element of risk for cardiovascular disease in adult life. [ABSTRACT FROM AUTHOR]

Hypoxia impairs metabolic functions by decreasing activity and expression of ATP-consuming processes. To separate hypoxia from systemic effects, we tested whether hypoxia at high altitude affects basal and PMA-stimulated leukocyte metabolism and how this compares to acute (15 min) and 24 h of in vitro hypoxia. Leukocytes were prepared at low altitude and ∼24 h after arrival at 4559 m. Mitochondrial oxygen consumption (JO2) was measured by respirometry, oxygen radicals by electron spin resonance spectroscopy, both at a Po2 = 100 mmHg (JO2,100) and 20 mmHg (JO2,20). Acute hypoxia of leukocytes decreased JO2 at low altitude. Exposure to high altitude decreased JO2,100, whereas JO2,20 was not affected. Acute hypoxia of low-altitude samples decreased the activity of complexes I, II, and III. At high altitude, activity of complexes I and III were decreased when measured in normoxia. Stimulation of leukocytes with PMA increased JO2,100 at low (twofold) and high altitude (five-fold). At both locations, PMA-stimulated JO2 was decreased by acute hypoxia. Basal and PMA-stimulated reactive oxygen species (ROS) production were unchanged at high altitude. Separate in vitro experiments performed at low altitude show that ∼75% of PMA-induced increase in JO2 was due to increased extra-mitochondrial JO2 (JO2,res; in the presence of rotenone and antimycin A). JO2,res was doubled by PMA. Acute hypoxia decreased basal JO2,res by ∼70% and PMA-stimulated JO2,res by about 50% in cells cultured in normoxia and hypoxia (1.5% O2; 24 h). Conversely, 24 h in vitro hypoxia decreased mitochondrial JO2,100 and JO2,20, extra-mitochondrial, basal, and PMA-stimulated JO2 were not affected. These results show that 24 h of high altitude but not 24 h in vitro hypoxia decreased basal leukocyte metabolism, whereas PMA-induced JO2 and ROS formation were not affected, indicating that prolonged high-altitude hypoxia impairs mitochondrial metabolism but does not impair respiratory burst. In contrast, acute hypoxia impairs respiratory burst at either altitude. [ABSTRACT FROM AUTHOR]

Protein levels and polymorphisms of p22phox have been suggested to modulate vascular NAD(P)H oxidase activity and vascular production of reactive oxygen species (ROS). We sought to determine whether increasing p22phox expression would alter vascular ROS production and hemodynamics by targeting p22phox expression to smooth muscle in transgenic (Tg) mice. Aortas of Tgp22smc mice had increased p22phox and Nox 1 protein levels and produced more superoxide anti H2O2. Surprisingly, endothelium-dependent relaxation and blood pressure in Tgp22smc mice were normal. Aortas of Tgp22smc mice produced twofold more nitric oxide (NO) at baseline and sevenfold more NO in response to calcium ionophore as detected by electron spin resonance. Western blot analysis revealed a twofold increase in endothelial NO synthase (eNOS) protein expression in Tgp22smc mice. Both eNOS expression and NO production were normalized by infusion of the glutathione peroxidase mimetic ebselen or by crossing Tgp22smc mice with mice overexpressing catalase. We have previously found that NO stimulares extracellular superoxide dismutase (ecSOD) expression in vascular smooth muscle. In keeping with this, aortic segments from Tgp22smc mice expressed twofold more ecSOD, and chronic treatment with the NOS inhibitor NG-nitro-L-arginine methyl ester normalized this, suggesting that NO regulates ecSOD protein expression in vivo. These data indicate that chronic oxidative stress caused by excessive H2O2 production evokes a compensatory response involving increased eNOS expression and NO production. NO in turn increases ecSOD protein expression and counterbalances increased ROS production leading to the maintenance of normal vascular function and hemodynamics. [ABSTRACT FROM AUTHOR]