Mycophenolate acid inhibits endothelial NAD(P)H oxidase activity and superoxide formation by a Rac1-dependent mechanism

Endothelial dysfunction precedes hypertension and atherosclerosis and predicts cardiac allograft vasculopathy and death in heart transplant recipients. Endothelial overproduction of reactive oxygen species, such as superoxide anions produced by NAD(P)H oxidase, induces endothelial dysfunction. Because immunosuppressive drugs have been associated with increased reactive oxygen species production and endothelial dysfunction, we sought to elucidate the underlying mechanisms. Reactive oxygen species, release of superoxide anions, and NAD(P)H oxidase activity were studied in human umbilical vein endothelial cells and in polymorphonuclear neutrophils. Gp91ds-tat was used to specifically block NAD(P)H oxidase. Transcriptional activation of different subunits of NAD(P)H oxidase was assessed by real-time RT-PCR. Rac1 subunit translocation and activation were studied by membrane fractionation and pull-down assays. Calcineurin inhibitors significantly increased endothelial superoxide anions production because of NAD(P)H oxidase, whereas mycophenolate acid (MPA) blocked it. MPA also attenuated the respiratory burst induced by neutrophil NAD(P)H oxidase. Because transcriptional activation of NAD(P)H oxidase was not affected, but addition of guanosine restored endothelial superoxide anions formation after MPA treatment, we speculate that the inhibitory effect of MPA was mediated by depletion of cellular guanosine triphosphate content. This prevented activation of Rac1 and, thus, of endothelial NAD(P)H oxidase. Because all heart transplant recipients are at risk for cardiac allograft vasculopathy development, these differential effects of immunosuppressants on endothelial oxidative stress should be considered in the choice of immunosuppressive drugs.

Rapid endocytosis of copper-zinc superoxide dismutase into human endothelial cells: role for its vascular activity

Cytosolic CuZn-SOD (SOD1) is a dimeric, carbohydrate-free enzyme with a molecular weight of about 32 kDa and also circulates in human blood plasma. Due to its molecular mass it has been believed that the enzyme cannot penetrate the cell membrane. Here we report that rapid endocytosis of FITC-CuZn-SOD into human endothelial cells occurs within 5 min. Moreover, relaxation of rat aortic rings in response to CuZn-SOD is associated with a lag time of 45-60 s and only observed in the presence of intact endothelial cells. The results indicate acute and rapid endothelial cell endocytosis of CuZn-SOD, possibly via activation of a receptor-mediated pathway. Intracellular uptake via endocytosis may contribute to the vascular effects of CuZn-SOD, including vasodilation, and is likely to play a role in regulation of vascular tone and diseases such as atherosclerosis.

Genetic polymorphisms of manganese-superoxide dismutase and glutathione-S-transferase in chronic alcoholic pancreatitis

Chronic alcohol consumption is a major risk factor for the development of chronic pancreatitis. However, chronic pancreatitis occurs only in a minority of heavy drinkers. This variability may be due to yet unidentified genetic factors. Several enzymes involved in the degradation of reactive oxidants and xenobiotics, such as glutathione-S-transferase P1 (GSTP1) and manganese-superoxide dismutase (MnSOD) reveal functional polymorphisms that affect the antioxidative capacity and may therefore modulate the development of chronic pancreatitis and long-term complications like endocrine and exocrine pancreatic insufficiency. Two functional polymorphisms of the MnSOD and the GSTP1 gene were assessed by polymerase chain reaction and restriction fragment length polymorphism in 165 patients with chronic alcoholic pancreatitis, 140 alcoholics without evidence of pancreatic disease and 160 healthy control subjects. The distribution of GSTP1 and MnSOD genotypes were in Hardy-Weinberg equilibrium in the total cohort. Genotype and allele frequencies for both genes were not statistically different between the three groups. Although genotype MnSOD Ala/Val was seemingly associated with the presence of exocrine pancreatic insufficiency, this subgroup was too small and the association statistically underpowered. None of the tested genotypes affected the development of endocrine pancreatic insufficiency. Polymorphisms of MnSOD and GSTP1 are not associated with chronic alcoholic pancreatitis. The present data emphasize the need for stringently designed candidate gene association studies with well-characterized cases and controls and sufficient statistical power to exclude chance observations.

Higher macrophage superoxide anion production in coronary artery disease (CAD) patients with Type D personality

BACKGROUND Type D personality (Type D) is an independent psychosocial risk factor for poor cardiac prognosis and increased mortality in patients with cardiovascular disease (CVD), but the involved mechanisms are poorly understood. Macrophages play a pivotal role in atherosclerosis, the process underlying coronary artery disease (CAD). We investigated macrophage superoxide anion production in production in CAD patients with and without Type D. METHODS AND RESULTS We studied 20 male CAD patients with Type D (M:66.7±9.9years) and 20 age-matched male CAD patients without Type D (M:67.7±8.5years). Type D was measured using the DS14 questionnaire with the two subscales 'negative affectivity' and 'social inhibition'. We assessed macrophage superoxide anion production using the WST-1 assay. All analyses were controlled for potential confounders. CAD patients with Type D showed higher superoxide anion production compared to CAD patients without Type D (F(1,38)=15.57, p<0.001). Complementary analyses using the Type D subscales 'negative affectivity' and 'social inhibition', and their interaction as continuous measures, showed that both Type D subscales (negative affectivity: (ß=0.48, p=0.002, R(2)=0.227); social inhibition: (ß=0.46, p=0.003, R(2)=0.208)) and their interaction (ß=0.36, p=0.022, R(2)=0.130) were associated with higher WST-1 reduction scores. Results remained significant when controlling for classical CVD risk factors (i.e. body mass index, mean arterial blood pressure), atherosclerosis severity (i.e. intima media thickness, presence of carotid plaques), and psychological factors (depressive symptom severity, chronic stress). CONCLUSIONS Our results indicate higher macrophage superoxide anion production in CAD patients with Type D compared to those without Type D. This may suggest a mechanism contributing to increased morbidity and mortality in CAD patients with Type D.

Paracrine factors secreted by endothelial progenitor cells prevent oxidative stress-induced apoptosis of mature endothelial cells

Endothelial progenitor cells (EPC) play a fundamental role in tissue regeneration and vascular repair. Current research suggests that EPC are more resistant to oxidative stress as compared to differentiated endothelial cells. Here we hypothesized that EPC not only possess the ability to protect themselves against oxidative stress but also confer this protection upon differentiated endothelial cells by release of paracrine factors. To test this hypothesis, HUVEC incubated with conditioned medium obtained from early EPC cultures (EPC-CM) were exposed to H2O2 to assess the accumulation of intracellular ROS, extent of apoptosis and endothelial cell functionality. Under oxidative stress conditions HUVEC treated with EPC-CM exhibited substantially lower levels of intracellular oxidative stress (0.2+/-0.02 vs. 0.4+/-0.03 relative fluorescence units, p<0.05) compared to control medium. Moreover, the incubation with EPC-CM elevated the expression level of antioxidant enzymes in HUVEC (catalase: 2.6+/-0.4; copper/zinc superoxide dismutase (Cu/ZnSOD): 1.6+/-0.1; manganese superoxide dismutase (MnSOD): 1.4+/-0.1-fold increase compared to control, all p<0.05). Furthermore, EPC-CM had the distinct potential to reverse the functional impairment of HUVEC as measured by their capability to form tubular structures in vitro. Finally, incubation of HUVEC with EPC-CM resulted in a significant reduction of apoptosis (0.34+/-0.01 vs. 1.52+/-0.12 relative fluorescence units, p<0.01) accompanied by an increased expression ratio of the anti/pro-apoptotic factors Bcl-2/Bax to 2.9+/-0.7-fold (compared to control, p<0.05). Most importantly, neutralization of selected cytokines such as VEGF, HGF, IL-8 and MMP-9 did not significantly reverse the cyto-protective effect of EPC-CM (p>0.05), suggesting that soluble factors secreted by EPC, possibly via broad synergistic actions, exert strong cyto-protective properties on differentiated endothelium through modulation of intracellular antioxidant defensive mechanisms and pro-survival signals.

A fluorescently labeled dendronized polymer-enzyme conjugate carrying multiple copies of two different types of active enzymes

A hybrid structure of a synthetic dendronized polymer, two different types of enzymes (superoxide dismutase and horseradish peroxidase), and a fluorescent dye (fluorescein) was synthesized. Thereby, a single polymer chain carried multiple copies of the two enzymes and the fluorescein. The entire attachment chemistry is based on UV/vis-quantifiable bis-aryl hydrazone bond formation that allows direct quantification of bound molecules: 60 superoxide dismutase, 120 horseradish peroxidase, and 20 fluorescein molecules on an average polymer chain of 2000 repeating units. To obtain other enzyme ratios the experimental conditions were altered accordingly. Moreover, it could be shown that both enzymes remained fully active and catalyzed a two-step cascade reaction.

Nitric oxide down-regulates the expression of the catalytic NADPH oxidase subunit Nox1 in rat renal mesangial cells

Glomerular mesangial cells can produce high amounts of nitric oxide (NO) and reactive oxygen species (ROS). Here we analyzed the impact of NO on the ROS-generating system, particularly on the NADPH oxidase Nox1. Nox1 mRNA and protein levels were markedly decreased by treatment of mesangial cells with the NO-releasing compound DETA-NO in a concentration- and time-dependent fashion. By altering the cGMP signaling system with different inhibitors or activators, we revealed that the effect of NO on Nox1 expression is at least in part mediated by cGMP. Analysis of a reporter construct comprising the 2547 bp of the nox1 promoter region revealed that a stimulatory effect of IL-1beta on nox1 transcription is counteracted by an inhibitory effect of IL-1beta-evoked endogenous NO formation. Moreover, pretreatment of mesangial cells with DETA-NO attenuated platelet-derived growth factor (PDGF)-BB or serum stimulated production of superoxide as assessed by real-time EPR spectroscopy and dichlorofluorescein formation. Transfection of mesangial cells with siRNAs directed against Nox1 and Nox4 revealed that inhibition of Nox1, but not Nox4 expression, is responsible for the reduced ROS formation by NO. Obviously, there exists a fine-tuned crosstalk between NO and ROS generating systems in the course of inflammatory diseases.

Exercise training in normobaric hypoxia in endurance runners. III. Muscular adjustments of selected gene transcripts

We hypothesized that specific muscular transcript level adaptations participate in the improvement of endurance performances following intermittent hypoxia training in endurance-trained subjects. Fifteen male high-level, long-distance runners integrated a modified living low-training high program comprising two weekly controlled training sessions performed at the second ventilatory threshold for 6 wk into their normal training schedule. The athletes were randomly assigned to either a normoxic (Nor) (inspired O2 fraction = 20.9%, n = 6) or a hypoxic group exercising under normobaric hypoxia (Hyp) (inspired O2 fraction = 14.5%, n = 9). Oxygen uptake and speed at second ventilatory threshold, maximal oxygen uptake (VO2 max), and time to exhaustion (Tlim) at constant load at VO2 max velocity in normoxia and muscular levels of selected mRNAs in biopsies were determined before and after training. VO2 max (+5%) and Tlim (+35%) increased specifically in the Hyp group. At the molecular level, mRNA concentrations of the hypoxia-inducible factor 1alpha (+104%), glucose transporter-4 (+32%), phosphofructokinase (+32%), peroxisome proliferator-activated receptor gamma coactivator 1alpha (+60%), citrate synthase (+28%), cytochrome oxidase 1 (+74%) and 4 (+36%), carbonic anhydrase-3 (+74%), and manganese superoxide dismutase (+44%) were significantly augmented in muscle after exercise training in Hyp only. Significant correlations were noted between muscular mRNA levels of monocarboxylate transporter-1, carbonic anhydrase-3, glucose transporter-4, and Tlim only in the group of athletes who trained in hypoxia (P < 0.05). Accordingly, the addition of short hypoxic stress to the regular endurance training protocol induces transcriptional adaptations in skeletal muscle of athletic subjects. Expressional adaptations involving redox regulation and glucose uptake are being recognized as a potential molecular pathway, resulting in improved endurance performance in hypoxia-trained subjects.

Decreased infarct size after focal cerebral ischemia in mice chronically infected with Toxoplasma gondii

To determine whether Toxoplasma gondii infection could modify biological phenomena associated with brain ischemia, we investigated the effect of permanent middle cerebral artery occlusion (MCAO) on neuronal survival, inflammation and redox state in chronically infected mice. Infected animals showed a 40% to 50% decrease of infarct size compared with non-infected littermates 1, 4 and 14 days after MCAO. The resistance of infected mice may be associated with increased basal levels of anti-inflammatory cytokines and/or a marked reduction of the MCAO-related brain induction of two pro-inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma (IFNgamma). In addition, potential anti-inflammatory/neuroprotective factors such as nerve growth factor, suppressor of cytokine signaling-3, superoxide dismutase activity, uncoupling protein-2 and glutathione (GSH) were upregulated in the brain of infected mice. Consistent with a role of GSH in central cytokine regulation, GSH depletion by diethyl maleate inhibited Toxoplasma gondii lesion resistance by increasing the proinflammatory cytokine IFNgamma brain levels. Overall, these findings indicate that chronic toxoplasmosis decisively influences both the inflammatory molecular events and outcome of cerebral ischemia.

Vitamin C deficiency in weanling guinea pigs: differential expression of oxidative stress and DNA repair in liver and brain

Neonates are particularly susceptible to malnutrition due to their limited reserves of micronutrients and their rapid growth. In the present study, we examined the effect of vitamin C deficiency on markers of oxidative stress in plasma, liver and brain of weanling guinea pigs. Vitamin C deficiency caused rapid and significant depletion of ascorbate (P < 0.001), tocopherols (P < 0.001) and glutathione (P < 0.001), and a decrease in superoxide dismutase activity (P = 0.005) in the liver, while protein oxidation was significantly increased (P = 0.011). No changes in lipid oxidation or oxidatively damaged DNA were observed in this tissue. In the brain, the pattern was markedly different. Of the measured antioxidants, only ascorbate was significantly depleted (P < 0.001), but in contrast to the liver, ascorbate oxidation (P = 0.034), lipid oxidation (P < 0.001), DNA oxidation (P = 0.13) and DNA incision repair (P = 0.014) were all increased, while protein oxidation decreased (P = 0.003). The results show that the selective preservation of brain ascorbate and induction of DNA repair in vitamin C-deficient weanling guinea pigs is not sufficient to prevent oxidative damage. Vitamin C deficiency may therefore be particularly adverse during the neonatal period.

Genetic and pharmacologic manipulation of oxidative stress after neonatal hypoxia-ischemia

Oxidative stress is a critical component of the injury response to hypoxia-ischemia (HI) in the neonatal brain, and this response is unique and at times paradoxical to that seen in the mature brain. Previously, we showed that copper-zinc superoxide-dismutase (SOD1) over-expression is not beneficial to the neonatal mouse brain with HI injury, unlike the adult brain with ischemic injury. However, glutathione peroxidase 1 (GPx1) over-expression is protective to the neonatal mouse brain with HI injury. To further test the hypothesis that an adequate supply of GPx is critical to protection from HI injury, we crossed SOD1 over-expressing mice (hSOD-tg) with GPx1 over-expressing mice (hGPx-tg). Resulting litters contained wild-type (wt), hGPx-tg, hSOD-tg and hybrid hGPx-tg/hSOD-tg pups, which were subjected to HI at P7. Confirming previous results, the hGPx-tg mice had reduced injury compared to both Wt and hSOD-tg littermates. Neonatal mice over-expressing both GPx1 and SOD1 also had less injury compared to wt or hSOD-tg alone. A result of oxidative stress after neonatal HI is a decrease in the concentration of reduced (i.e. antioxidant-active) glutathione (GSH). In this study, we tested the effect of systemic administration of alpha-lipoic acid on levels of GSH in the cortex after HI. Although GSH levels were restored by 24h after HI, injury was not reduced compared to vehicle-treated mice. We also tested two other pharmacological approaches to reducing oxidative stress in hSOD-tg and wild-type littermates. Both the specific inhibitor of neuronal nitric oxide synthase, 7-nitroindazole (7NI), and the spin-trapping agent alpha-phenyl-tert-butyl-nitrone (PBN) did not reduce HI injury, however. Taken together, these results imply that H2O2 is a critical component of neonatal HI injury, and GPx1 plays an important role in the defense against this H2O2 and is thereby neuroprotective.

Manipulation of antioxidant pathways in neonatal murine brain

To assess the role of brain antioxidant capacity in the pathogenesis of neonatal hypoxic-ischemic brain injury, we measured the activity of glutathione peroxidase (GPX) in both human-superoxide dismutase-1 (hSOD1) and human-GPX1 overexpressing transgenic (Tg) mice after neonatal hypoxia-ischemia (HI). We have previously shown that mice that overexpress the hSOD1 gene are more injured than their wild-type (WT) littermates after HI, and that H(2)O(2) accumulates in HI hSOD1-Tg hippocampus. We hypothesized that lower GPX activity is responsible for the accumulation of H(2)O(2). Therefore, increasing the activity of this enzyme through gene manipulation should be protective. We show that brains of hGPX1-Tg mice, in contrast to those of hSOD-Tg, have less injury after HI than WT littermates: hGPX1-Tg, median injury score = 8 (range, 0-24) versus WT, median injury score = 17 (range, 2-24), p < 0.01. GPX activity in hSOD1-Tg mice, 2 h and 24 h after HI, showed a delayed and bilateral decline in the cortex 24 h after HI (36.0 +/- 1.2 U/mg in naive hSOD1-Tg versus 29.1 +/- 1.7 U/mg in HI cortex and 29.2 +/- 2.0 for hypoxic cortex, p < 0.006). On the other hand, GPX activity in hGPX1-Tg after HI showed a significant increase by 24 h in the cortex ipsilateral to the injury (48.5 +/- 5.2 U/mg, compared with 37.2 +/- 1.5 U/mg in naive hGPX1-Tg cortex, p < 0.008). These findings support the hypothesis that the immature brain has limited GPX activity and is more susceptible to oxidative damage and may explain the paradoxical effect seen in ischemic neonatal brain when SOD1 is overexpressed.

Fructose-1,6-bisphosphate preserves intracellular glutathione and protects cortical neurons against oxidative stress

Fructose-1,6-bisphosphate (FBP), an endogenous intermediate of glycolysis, protects the brain against ischemia-reperfusion injury. The mechanisms of FBP protection after cerebral ischemia are not well understood. The current study was undertaken to determine whether FBP protects primary neurons against hypoxia and oxidative stress by preserving reduced glutathione (GSH). Cultures of pure cortical neurons were subjected to oxygen deprivation, a donor of nitric oxide and superoxide radicals (3-morpholinosydnonimine), an inhibitor of glutathione synthesis (L-buthionine-sulfoximine) or glutathione reductase (1,3-bis(2-chloroethyl)-1-nitrosourea) in the presence or absence of FBP (3.5 mM). Neuronal viability was determined using an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. FBP protected neurons against hypoxia-reoxygenation and oxidative stress under conditions of compromised GSH metabolism. The efficacy of FBP depended on duration of hypoxia and was associated with higher intracellular GSH concentration, an effect partly mediated via increased glutathione reductase activity.

Cerebral vasculature is the major target of oxidative protein alterations in bacterial meningitis

We have previously shown that antioxidants such as a-phenyl-tert-butyl nitrone or N-acetylcysteine attenuate cortical neuronal injury in infant rats with bacterial meningitis, suggesting that oxidative alterations play an important role in this disease. However, the precise mechanism(s) by which antioxidants inhibit this injury remain(s) unclear. We therefore studied the extent and location of protein oxidation in the brain using various biochemical and immunochemical methods. In cortical parenchyma, a trend for increased protein carbonyls was not evident until 21 hours after infection and the activity of glutamine synthetase (another index of protein oxidation) remained unchanged. Consistent with these results, there was no evidence for oxidative alterations in the cortex by various immunohistochemical methods even in cortical lesions. In contrast, there was a marked increase in carbonyls, 4-hydroxynonenal protein adducts and manganese superoxide dismutase in the cerebral vasculature. Elevated lipid peroxidation was also observed in cerebrospinal fluid and occasionally in the hippocampus. All of these oxidative alterations were inhibited by treatment of infected animals with N-acetylcysteine or alpha-phenyl-tert-butyl nitrone. Because N-acetylcysteine does not readily cross the blood-brain barrier and has no effect on the loss of endogenous brain antioxidants, its neuroprotective effect is likely based on extraparenchymal action such as inhibition of vascular oxidative alterations.