Homocysteine stimulates NADPH oxidase-mediated superoxide production leading to endothelial dysfunction in rats

Elevation of blood homocysteine (Hcy) levels (hyperhomocysteinemia) is a risk factor for cardiovascular disorders. We previously reported that oxidative stress contributed to Hcy-induced inflammatory response in vascular cells. In this study, we investigated whether NADPH oxidase was involved in Hcy-induced superoxide anion accumulation in the aorta, which leads to endothelial dysfunction during hyperhomocysteinemia. Hyperhomocysteinemia was induced in rats fed a high-methionine diet. NADPH oxidase activity and the levels of superoxide and peroxynitrite were markedly increased in aortas isolated from hyperhomocysteinemic rats. Expression of the NADPH oxidase subunit p22 phox increased significantly in these aortas. Administration of an NADPH oxidase inhibitor (apocynin) not only attenuated aortic superoxide and peroxynitrite to control levels but also restored endothelium-dependent relaxation in the aortas of hyperhomocysteinemic rats. Transfection of human endothelial cells or vascular smooth muscle cells with p22 phox siRNA to inhibit NADPH oxidase activation effectively abolished Hcy-induced superoxide anion production, thus indicating the direct involvement of NADPH oxidase in elevated superoxide generation in vascular cells. Taken together, these results suggest that Hcy-stimulated superoxide anion production in the vascular wall is mediated through the activation of NADPH oxidase, which leads to endothelial dysfunction during hyperhomocysteinemia.     

Normobaric hyperoxia inhibits NADPH oxidase-mediated matrix metalloproteinase-9 induction in cerebral microvessels in experimental stroke

Matrix metalloproteinase-9 (MMP-9) and NADPH oxidase contribute to blood-brain barrier (BBB) disruption after ischemic stroke. We have previously shown that normobaric hyperoxia (NBO) treatment reduces MMP-9 and oxygen free radical generation in ischemic brain. In this study, we tested the hypothesis that NBO protects the BBB through inhibiting NADPH oxidase-mediated MMP-9 induction in transient focal cerebral ischemia. Male Sprague-Dawley rats (n = 69) were given NBO (95% O2) or normoxia (21% O2) during 90-min filament occlusion of the middle cerebral artery. Cerebral microvessels were isolated for analyzing MMP-9 and NADPH oxidase. BBB damage was non-invasively quantified with magnetic resonance imaging. In normoxic rats, both NADPH oxidase catalytic subunit gp91(phox) and MMP-9 expression were up-regulated in ischemic hemispheric microvessels after 90-min middle cerebral artery occlusion with 22.5 h reperfusion. Inhibition of NADPH oxidase with apocynin reduced the MMP-9 increase, indicating a causal link between NADPH oxidase-derived superoxide and MMP-9 induction. NBO treatment inhibited gp91(phox) expression, NADPH oxidase activity, and MMP-9 induction, which led to significantly less BBB damage and brain edema in the ischemic brain. These results suggest that gp91(phox) containing NADPH oxidase plays an important role in MMP-9 induction in ischemic BBB microvasculature, and that NBO treatment may attenuate MMP-9 induction and brain edema through inhibiting NADPH oxidase after transient cerebral ischemia.     

Effects of in vitro and in vivo supplementation with zinc on superoxide anion production in leukocytes

The effects of zinc on the rate of production of bactericidal O2- of polymorphonuclear leukocytes (PMN) in response to three different types of stimulating agents (serum-treated zymosan (STZ), Con A, and myristate) were studied. The percentage reduction of O2- production of PMN stimulated by STZ, Con A, and myristate were all reduced in response to Zn, irregardless of whether Zn was added to the reaction mixture immediately before SZT addition or following a prior 20 min. incubation of PMN in the presence of Zn. However, when Zn was introduced intraperitonially into guinea pigs before the collection of PMN from the animal, zinc treatment produced inhibition only in STZ-activated PMN; it produced no effect in O2- production of PMN stimulated by myristate, and it further augmented the O2- production stimulated by Con A.     

Detergent-amplified chemiluminescence of lucigenin for determination of superoxide anion production by NADPH oxidase and xanthine oxidase

The detergent-induced amplification of lucigenin-dependent chemiluminescence of O2-, generated by xanthine oxidase or microsomal NADPH oxidase was studied. An assay system is described which is at least 10 times more sensitive than normal lucigenin-dependent chemiluminescence due to the amplification by high concentrations of octylphenylpolyethylene glycol (Triton X-100). Compared to the superoxide dismutase-sensitive reduction of acetylated cytochrome c, a 3750-fold lower amount of microsomal protein was necessary to produce an O2- signal 10-fold above the background. In contrast to cytochrome c reduction, detergent-amplified chemiluminescence of lucigenin was completely inhibited by superoxide dismutase and therefore more selective for O2-. The membrane-bound and Triton X-100-solubilized NADPH oxidase from microsomes of macrophages was activated by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and inhibited by Ca2+ and sodium dodecyl sulfate. The membrane-bound enzyme showed a Km value of 1.35 microM, which decreased to 0.95 microM after the addition of 12% (g/g) Triton X-100. The Km and Vmax values of soluble xanthine oxidase were not influenced by Triton X-100, indicating that the enzyme activities were not impaired by the high concentrations of detergent.     

Low-grade chronic inflammation and superoxide anion production by NADPH oxidase are the main determinants of physical frailty in older adults

Abstract

Background. Physical performance measured by gait speed is being recognized as a major instrument for clinical evaluation in older adults, because it predicts physical frailty, loss of autonomy, hospitalization and decreased survival. Low-grade chronic inflammation and oxidative stress, mediated partly by the superoxide anion produced by NADPH oxidase, are closely linked and could be involved in age-related physical decline. Objective. To determine whether slow gait speed is associated with superoxide anion overproduction by NADPH oxidase and low-grade chronic inflammation. Design and setting. Observational study among the 280 elderly of an ambulatory geriatric care unit (191 women, 89 men, 79.9 ± 6.1 years old). Methods. Gait speed was evaluated by walking at self-chosen usual pace. Usual gait speed < 0.8 m/s was defined as slow gait speed. Superoxide anion production was evaluated using a lucigenin-based chemiluminescence method. Inflammation was evaluated by CRP, fibrinogen and leukocyte count. Results. Among the 280 participants, 179 (63.9%) walked with a gait speed < 0.8 m/s (slow walkers) and 101 (36.1%) with a gait speed ≥ 0.8 m/s. Superoxide production and inflammation markers, such as fibrinogen, were more important in slow walkers (p = 0.004 and p = 0.006, respectively). In multivariate analysis, superoxide anion overproduction and fibrinogen were independently associated with physical frailty assessed by slow gait speed (p = 0.028 and p = 0.007, respectively). Conclusion. Physical frailty in older people is associated with superoxide anion overproduction by NADPH oxidase and low-grade chronic inflammation.     

Folic acid supplementation delays atherosclerotic lesion development in apoE-deficient mice

Folic acid is a vitamin that when used as a dietary supplementation can improve endothelial function. To assess the effect of folic acid on the development of atherosclerosis, male apolipoprotein E-deficient mice fed a standard chow diet received either water (control group) or an aqueous solution of folic acid that provided a dose of 75 microg/kg/day, for ten weeks. At the time of sacrifice, blood was drawn and the heart removed. The study measured plasma homocysteine, lipids, lipoproteins, low-density lipoprotein (LDL) oxidation, isoprostane, paraoxonase, and apolipoproteins, and aortic atherosclerotic areas. In folic acid-treated animals, total cholesterol, mainly carried in very low-density and low-density lipoproteins, increased significantly, and homocysteine, HDL cholesterol, paraoxonase, and triglyceride levels did not change significantly. Plasma isoprostane and apolipoprotein (apo) B levels decreased. The resistance of LDL to oxidization and plasma apoA-I and apoA-IV levels increased with a concomitant decrease in the area of atherosclerotic lesions. The administration of folic acid decreased atherosclerotic lesions independently of plasma homocysteine and cholesterol levels, but was associated with plasma levels of apolipoproteins A-I, A-IV and B, and decreased oxidative stress.     

Lipoxygenase-mediated production of superoxide anion in senescing plant tissue

Lipoxygenase activity and superoxide (O^.?₂) production by microsomal membranes and cytosol from bean cotyledons increased in parallel as senescence progressed. Superoxide production was heat denaturable and dependent on the availability of linoleate, the substrate for lipoxygenase. The specific inhibitor of lipoxygenase, U28938, caused a parallel reduction in enzyme activity and the formation of O^?₂. These observations demonstrate that lipoxygenase activity mediates the formation of superoxide anion, and support the contention that membrane senescence is attributable to a sequence of reactions in which lipasederived fatty acids are utilized by lipoxygenase to generate O^?₂ and hydroperoxides.     

Folic acid supplementation and neural tube defect recurrence prevention

BACKGROUND: It is well established that women who have had a pregnancy affected by a neural tube defect (NTD) have an elevated risk of a subsequent NTD-affected pregnancy and that a high dose (4 mg/day) of folic acid taken around the time of conception prevents most recurrences of NTDs. METHODS: We reviewed the literature to identify studies that quantify the reduction in risk if women with a prior-NTD affected pregnancy consistently take folic acid before and during a subsequent pregnancy and the effectiveness of NTD recurrence prevention programs in increasing the percentage of women who consistently consume folic acid supplements. RESULTS: A meta-analysis of randomized trials of folic acid for the prevention of recurrent NTDs indicates a 69% reduction in recurrence risk if analyzed on an intention-to-treat basis and an 87% reduction among those women who took supplements prior to the beginning of pregnancy. Observational studies report reductions in recurrence risk of 85% to 100% among women taking folic acid prior to subsequent pregnancies. The percentage of women who take folic acid prior to a subsequent pregnancy has been reported to vary from 33% to 85%, varying with the demographic background and the intensity of folic acid counseling efforts. CONCLUSIONS: Targeted folic acid information and counseling provided to women with an NTD-affected pregnancy has been demonstrated to substantially reduce the risk of recurrent NTDs and is feasible to implement on a public health basis.     

Adverse effects of the classic antioxidant uric acid in adipocytes: NADPH oxidase-mediated oxidative/nitrosative stress

Uric acid is considered a major antioxidant in human blood that may protect against aging and oxidative stress. Despite its proposed protective properties, elevated levels of uric acid are commonly associated with increased risk for cardiovascular disease and mortality. Furthermore, recent experimental studies suggest that uric acid may have a causal role in hypertension and metabolic syndrome. All these conditions are thought to be mediated by oxidative stress. In this study we demonstrate that differentiation of cultured mouse adipocytes is associated with increased production of reactive oxygen species (ROS) and uptake of uric acid. Soluble uric acid stimulated an increase in NADPH oxidase activity and ROS production in mature adipocytes but not in preadipocytes. The stimulation of NADPH oxidase-dependent ROS by uric acid resulted in activation of MAP kinases p38 and ERK1/2, a decrease in nitric oxide bioavailability, and an increase in protein nitrosylation and lipid oxidation. Collectively, our results suggest that hyperuricemia induces redox-dependent signaling and oxidative stress in adipocytes. Since oxidative stress in the adipose tissue has recently been recognized as a major cause of insulin resistance and cardiovascular disease, hyperuricemia-induced alterations in oxidative homeostasis in the adipose tissue might play an important role in these derangements.     

Folic acid supplementation dysregulates gene expression in lymphoblastoid cells--implications in nutrition

For over a decade, folic acid (FA) supplementation has been widely prescribed to pregnant women to prevent neural tube closure defects in newborns. Although neural tube closure occurs within the first trimester, high doses of FA are given throughout pregnancy, the physiological consequences of which are unknown. FA can cause epigenetic modification of the cytosine residues in the CpG dinucleotide, thereby affecting gene expression. Dysregulation of crucial gene expression during gestational development may have lifelong adverse effects or lead to neurodevelopmental defects, such as autism. We have investigated the effect of FA supplementation on gene expression in lymphoblastoid cells by whole-genome expression microarrays. The results showed that high FA caused dysregulation by ?four-fold up or down to more than 1000 genes, including many imprinted genes. The aberrant expression of three genes (FMR1, GPR37L1, TSSK3) was confirmed by Western blot analyses. The level of altered gene expression changed in an FA concentration-dependent manner. We found significant dysregulation in gene expression at concentrations as low as 15 ng/ml, a level that is lower than what has been achieved in the blood through FA fortification guidelines. We found evidence of aberrant promoter methylation in the CpG island of the TSSK3 gene. Excessive FA supplementation may require careful monitoring in women who are planning for, or are in the early stages of pregnancy. Aberrant expression of genes during early brain development may have an impact on behavioural characteristics.     

Polyamines inhibit NADPH oxidase-mediated superoxide generation and putrescine prevents programmed cell death induced by polyamine oxidase-generated hydrogen peroxide

Our previous results indicate that during protoplast isolation an oxidative burst occurs [A.K. Papadakis and KA Roubelakis-Angelakis (1999) Plant Physiol 127:197–205] and that suppression of totipotency is correlated with reduced antioxidant activity and low redox state [A.K. Papadakis et al. (2001b) Plant Physiol 126:434–444]. Polyamines are known to affect cell development and to act as antioxidants. Polyamines applied during isolation of tobacco (Nicotiana tabacum L.) protoplasts reduced the accumulation of O₂^·– but not that of H₂O₂. This antioxidant effect is probably due to the inhibition of microsomal membrane NADPH oxidase, which occurred in a concentration-dependent manner, with spermine exerting the highest inhibitory effect. However, during protoplast culture, polyamine oxidase activity increased severalfold in spermidine- and spermine-treated protoplasts, concomitant with H₂O₂ titers. A cell death program was executed in untreated protoplasts, as documented by membrane malfunction, induced DNase activity, DNA fragmentation and a positive TUNEL reaction. Protoplast cell death was prevented in protoplasts treated with putrescine, but not by treatment with spermidine or spermine, which rather had the opposite effect. The data presented suggest that PAs may be implicated in the expression of plant protoplast totipotency.     

Diacylglycerol accumulation and superoxide anion production in stimulated human neutrophils

Exogenous diacylglycerols stimulate neutrophil superoxide anion production, suggesting that endogenous diacylglycerols may function as second messengers for this biological response. We have measured the diacylglycerol mass in human neutrophils stimulated by fMet-Leu-Phe, ionomycin, and concanavalin A and have correlated the kinetics and magnitude of the diacylglycerol response with those for superoxide anion production. For each stimulus, no increase in diacylglycerol mass was detected prior to the onset of superoxide anion generation. However, large sustained increases in diacylglycerol concentration (260-2000% of basal levels) occurred in parallel with the rise in superoxide anion. The cessation or continuation of diacylglycerol accumulation and superoxide anion production also correlated. The diacylglycerol response was proportional to the stimulus concentration and correlated with the concentration dependence for superoxide anion. Pretreatment of neutrophils with cytochalasin B enhanced both superoxide anion and diacylglycerol responses with all three stimuli. These data support the hypothesis that diacylglycerol functions as a modulator of superoxide anion generation causing a sustained or augmented respiratory burst.     

Inhibition of superoxide anion production by extracellular acidification in neutrophils

Extracellular acidification inhibited formyl-Met-Leu-Phe- or C5a-induced superoxide anion (O₂⁻) production in differentiated HL-60 neutrophil-like cells and human neutrophils. A cAMP-increasing agonist, prostaglandin E₁, also inhibited the formyl peptide-induced O₂⁻ production. The inhibitory action on the O₂⁻ production by extracellular acidic pH was associated with cAMP accumulation and partly attenuated by H89, a protein kinase A inhibitor. A significant amount of mRNAs for T-cell death-associated gene 8 (TDAG8) and other proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1)-family receptors is expressed in these cells. These results suggest that cAMP/protein kinase A, possibly through proton-sensing G-protein-coupled receptors, may be involved in extracellular acidic pH-induced inhibition of O₂⁻ production.