Abstract-Glucocorticoid (GC) excess often elicits serious adverse effects on the vascular system, such as hypertension and atherosclerosis, and effective prophylaxis for these complications is limited. We sought to reveal the mechanism underlying GC-induced vascular complications. Responses in forearm blood flow to reactive hyperemia in 20 GC-treated patients were significantly decreased to 43Ϯ8.9% (meanϮSEM) from the values obtained before GC therapy (130Ϯ14%). An administration of vitamin C almost normalized blood flow responses. In human umbilical vein endothelial cells (HUVECs), production of hydrogen peroxide was increased up to 166.5Ϯ3.3% of control values by 10 Ϫ7 mol/L dexamethasone (DEX) treatment (PϽ0.01). Concomitant with DEX-induced hydrogen peroxide production, intracellular amounts of peroxynitrite significantly increased and those of nitric oxide (NO) decreased, respectively (PϽ0.01). Immunoblotting analysis using anti-nitrotyrosine antibody showed that peroxynitrite formation was increased in DEX-treated HUVECs. Using inhibitors against metabolic pathways for generation of reactive oxygen species (ROS), we identified that the major production sources of ROS by DEX treatment were mitochondrial electron transport chain, NAD(P)H oxidase, and xanthine oxidase. These findings suggest that GC excess causes overproduction of ROS and thereby perturbs NO availability in the vascular endothelium, leading to vascular complications in patients with GC excess. Key Words: glucocorticoid Ⅲ reactive oxygen species Ⅲ nitric oxide Ⅲ vascular endothelial function G lucocorticoid (GC) has being used widely for the treatment of patients with various disorders including autoimmune diseases, allergic diseases, and lymphoproliferative disorders. It has been well known, however, that GC therapy using prednisolone, methylprednisolone, or dexamethasone (DEX) is often limited by several adverse reactions associated with GC excess. 1,2 GC excess exhibits a variety of symptoms and signs, including truncal obesity with moon face, striae, hirsutism, cataract, osteoporosis, myopathy, diabetes mellitus, immunosuppression, and cardiovascular disorders such as hypertension and atherosclerosis. 3 Among these, cardiovascular complications are one of the important factors for predicting the morbidity and mortality of patients with GC excess. 3 Plasma volume expansion due to sodium retention plays a minor role, 1,2,4 and increased peripheral vascular resistance due in part to an increased pressor response to catecholamines and angiotensin II is shown to play a major role in the pathogenesis of hypertension induced by GC excess. 1,2,5 However, the molecular mechanism whereby GC excess causes the increase in vascular resistance remains unclear.Vascular endothelial cells regulate vascular tone through the release of a variety of relaxing and contracting factors that modulate the contractile activity of vascular smooth muscle cells. 6,7 Nitric oxide (NO), an endothelial cell-derived relaxing factor, is thought to be the most important vaso...

Abstract.We immunohistochemically investigated the localization of activin A and follistatin in various human tissues with specific antibodies to recombinant human (rh-) activin A and rh-follistatin.

A sensitive and specific immunoradiometric assay for follistatin was developed using antifollistatin mouse monoclonal and rabbit polyclonal antibodies. The sensitivity of the assay was 0.5 micrograms/L, and cross-reactivities with recombinant human activin A and bovine inhibin were less than 0.1%. The intra- and interassay coefficients of variation were less than 10%, and the recovery rate was about 90% in human serum. The addition of activin A to the same sample resulted in a minimal influence on follistatin recovery, indicating that this assay system can measure the total level of activin-bound and unbound follistatin. Gel filtration analysis of human serum showed that the majority of immunoreactivity was eluted in a larger molecular size position than that of free follistatin, suggesting that the large part of follistatin is bound to other proteins, presumably activins, in serum. Using this assay, immunoreactive follistatin levels in various biological fluids and human sera were examined. The dose-response curves of porcine follicular and amniotic fluids were parallel to the standard curve, and porcine follicular fluid contained extremely high follistatin immunoreactivity (5.6 mg/L). The serum follistatin level in normal human volunteers was 13.3 +/- 4.7 micrograms/L (mean +/- SD; n = 60), with a tendency to increase gradually with age. On the other hand, the serum follistatin level was remarkably elevated in pregnant women (62.7 +/- 35.3 micrograms/L; n = 57), with a positive correlation with weeks of pregnancy. These data indicated that circulating immunoreactive follistatin is detectable in human serum, and the levels vary with physiological conditions such as aging and pregnancy.

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The time-of-flight method has been employed to investigate the type and energy of high-energy neutral atoms bombarding the substrates in films of ZnO and BaTiO3 prepared by both DC planar magnetron sputtering and conventional DC diode sputtering. The high-energy neutral atoms are found to be oxygen atoms. The dependence of the flux of high-energy neutral oxygen atoms and negative oxygen ions on the pressure has also been measured for the planar magnetron sputtering of ZnO. It is shown that the high-energy particles bombarding the substrate are composed of neutral oxygen atoms at higher gas pressures ranging above 0.01 Torr, but negative oxygen ions are comparable with neutral oxygen atoms at pressures of the order of 10-3 Torr. The production mechanism of these energetic oxygen species is discussed.

Dips were observed in the distribution curves of the deposition rates of ZnO films prepared both by planar diode and by planar magnetron sputtering. These dips are ascribed to high-energy neutral atoms bombarding the films. The influence of high-energy neutral atoms on the mixed orientation of ZnO film is also investigated in planar diode sputtering. In planar magnetron sputtering, which gives highly [00·2] oriented ZnO films, the correlation between the degree of c-axis orientation and the flux of high-energy neutral atoms is examined. It is found that high-energy neutral atoms should be considered when trying to obtain highly-oriented ZnO films.

Articles you may be interested inElectrical properties of indium-tin oxide films deposited on nonheated substrates using a planar-magnetron sputtering system and a facing-targets sputtering system J. Vac. Sci. Technol. A 24, 65 (2006); 10.1116/1.2134711Indium-tin oxide films radio frequency sputtered from specially formulated high density indium-tin oxide targets J. Vac. Sci. Technol. A 9, 1193 (1991); 10.1116/1.577601 Transparent conducting zinc oxide and indium-tin oxide films prepared by modified reactive planar magnetron sputtering J.Energetic negative ions and neutral atoms were simultaneously observed using a time-of-flight apparatus during indium-tin oxide lITO) film preparation by conventional planar magnetron sputtering of an ITO target. The sputtering was performed in pure Ar gas and compared with ZnO:Al sputtering. Energetic particles such as 0-and O 2 ions and neutral 0 atoms were detected in L~e time-of-flight spectra, although the flux intensity of these particles was much less than that found in the ZnO sputtering. It was also found that other particles exist at the same time, which were thought to be reilected Ar atoms or 0 3 ions from the target surface. However, the flux intensity of these particles was found to be less than that of the energetic 0 -ions and 0 atoms. The strong energetic particles, which primarily bombarded the substrate during the ITO sputtering were found to be energetic 0ions and a atoms.

We developed an assay system for measuring free follistatin by using an anti-follistatin mouse monoclonal antibody and [125I]activin A. The sensitivity of this assay was 0.5 microgram/l and cross-reactivities with inhibin, luteinizing hormone, follicle-stimulating hormone and growth hormone were all less than 0.5%. The dose-response curves of human sera and follicular fluid were parallel to the standard curve, and the follicular fluid contained a large amount of follistatin (6.4 +/- 0.5 mg/l, mean +/- SEM; N = 13). The within- and between-assay coefficients of variation calculated from the analysis of serum samples of four different concentrations were 3.3-7.8% and 3.9-11.0%, respectively. The recovery rates of free follistatin at five different doses were 86.4 - 102.4%. When activin A was added to the same sample, free follistatin recovery rate declined dose-dependently. Gel filtration analyses of human serum and follicular fluid resulted in a single peak corresponding to authentic follistatin. Using this assay, free follistatin concentrations in sera were measured in normal, pregnant and diseased subjects. The free follistatin level in serum of normal adults was 3.5 +/- 0.2 micrograms/l (N = 60), which was significantly elevated in pregnant women (16.7 +/- 1.3 micrograms/l, N = 56), and in patients with chronic liver disease (8.1 +/- 1.1 micrograms/l, N = 20), chronic renal failure (6.7 +/- 0.9 micrograms/l, N = 42), advanced solid cancer (8.5 +/- 1.0 micrograms/l, N = 39) and hematological malignancies (6.8 +/- 1.0 micrograms/l, N = 18). These data indicated that the free follistatin concentration in serum is detectable and varies during pregnancy and in various diseased states.