EZETIMIBE PROTECTS THP-1 CELLS FROM ISCHEMIA-REPERFUSION INJURY REDUCING OXIDATIVE STRESS AND UP-REGULATING NRF2/ ARE GENE EXPRESSION

Background and Aims: We demonstrated that physical training, characterized by repeated ischemia-reperfusion (I-R) episodes (ischemic conditioning, IC), protects circulating cells from peripheral artery disease (PAD) patients against ischemic harms by reducing oxidative stress (OS) and by up-regulating nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway expression. Ezetimibe (Eze) has been shown to alleviate OS enhancing Nrf2 nuclear translocation in an AMPK/p62-dependent manner. In a cellular I-R and IC model, we aimed to investigate: 1) the effect of Eze on OS and Nrf2/ARE gene expression 2) whether Eze could have a synergistic effect on IC. Methods: THP-1 cells were treated with or without Eze (50mM) overnight, then subjected to 1 or 6 repetitive I-R cycles using EVOS FL Auto Imaging System. Reactive oxygen species (ROS) formation was evaluated with DCF in cytofluorimetry. Nrf2/ARE and p62 gene expression were evaluated by RT-PCR and western blotting. Results: When THP-1 cells were exposed to 1 I-R cycle, the preincubation with Eze significantly reduced ROS formation (p<0.01) and up-regulated Nrf2/ARE pathway expression and p62 phosphorylation (p<0.001). Multiple I-R cycles, acting as IC, significantly reduced ROS formation and upregulated Nrf2/ARE gene expression (p<0.001); in these conditions, Eze preincubation was able not only to almost abolish ROS formation (p<0.01) but also further up-regulate Nrf2/ARE expression. Conclusions: In our I-R model, Eze not only restores I-R-induced oxidative damages through Nrf2/ARE signaling up-regulation but also has a synergistic effect on IC. This new \u201cpleiotropic\u201d effect, if confirmed in vivo, may strengthen the use of Eze in PAD patien

Serum oxidative stress-induced repression of Nrf2 and GSH depletion: a mechanism potentially involved in endothelial dysfunction of young smokers

AbstractBackground: Although oxidative stress plays a major role in endothelial dysfunction (ED), the role of glutathione (GSH), ofnuclear erythroid-related factor 2 (Nrf2) and of related antioxidant genes (ARE) are yet unknown. In this study we combined an in vivo with an in vitro model to assess whether cigarette smoking affects flow-mediated vasodilation (FMD), GSHconcentrations and the Nrf2/ARE pathway in human umbilical vein endothelial cells (HUVECs).Methods and Results: 52 healthy subjects (26 non-smokers and 26 heavy smokers) were enrolled in this study. In smokerswe demonstrated increased oxidative stress, i.e., reduced concentrations of GSH and increased concentrations of oxidationproducts of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC) in serum and inperipheral blood mononuclear cells (PBMC), used as in vivo surrogates of endothelial cells. Moreover we showedimpairment of FMD in smokers and a positive correlation with the concentration of GSH in PBMC of all subjects. In HUVECsexposed to smokers\u2019 serum but not to non-smokers\u2019 serum we found that oxidative stress increased, whereas nitric oxideand GSH concentrations decreased; interestingly the expression of Nrf2, of heme oxygenase-1 (HO-1) and of glutamatecysteineligase catalytic (GCLC) subunit, the rate-limiting step of synthesis of GSH, was decreased. To test the hypothesisthat the increased oxidative stress in smokers may have a causal role in the repression of Nrf2/ARE pathway, we exposedHUVECs to increasing concentrations of oxPAPC and found that at the highest concentration (similar to that found insmokers\u2019 serum) the expression of Nrf2/ARE pathway was reduced. The knockdown of Nrf2 was associated to a significantreduction of HO-1 and GCLC expression induced by oxPAPC in ECs.Conclusions: In young smokers with ED a novel further consequence of increased oxidative stress is a repression of Nrf2/ARE pathway leading to GSH depletion

Nebivolol treatment reduces serum levels of asymmetric dimethylarginine and improves endothelial dysfunction in essential hypertensive patients.

This study was conducted to evaluate (i) the effect of nebivolol, a selective beta1-adrenergic receptor antagonist, on plasma concentration of asymmetric dimethylarginine (ADMA) and on flow-mediated dilation (FMD) in essential hypertensive patients; (ii) the effect of serum derived from the treated hypertensive patients on ADMA and on dimethylarginine dimethylaminohydrolase 2 (DDAH2), the enzyme that selectively degrades ADMA, in human umbilical vein endothelial cells (HUVECs).Forty healthy subjects and 40 matched essential hypertensive patients treated with atenolol and nebivolol according to a double-blind, randomized design participated in the study. Evaluation of brachial artery (BA) reactivity was performed by a longitudinal B-mode scan of the right BA. ADMA and L-arginine were measured by high-performance liquid chromatography. DDAH2 expression and endothelial nitric oxide synthase activity (eNOS) were also evaluated in HUVECs.ADMA levels were significantly decreased and FMD increased only in patients receiving nebivolol (P < 0.01). Furthermore, in nebivolol group, we found a significant correlation between changes in ADMA levels and changes in FMD (P < 0.01). Sera derived from patients treated with nebivolol but not with atenolol decreased ADMA and increased DDAH2 expression and eNOS activity (P < 0.001) in HUVECs.The results of this study demonstrate that the improvement of endothelial dysfunction induced by nebivolol in hypertensive patients may be related to its effect on circulating ADMA levels. Although the mechanism by which nebivolol reduces circulating ADMA in hypertensive patients remains unclear, our ex vivo results suggest that the upregulation of DDAH2 expression may have a role

White blood cells, FeNO, glutathione, 8-oxodG and 8-isoprostane in respiratory diseases

Inflammation and oxidative stress (OS) play an important role in pathogenesis of respiratory diseases. Such biomarkers as fractional exhaled nitric oxide (FeNO), white blood cells, glutathione, urinary 8-oxodG and 8-isoprostane can serve in evaluating clinical course of the disease. We aimed at estimating the association of biomarkers of OS and inflammation with current asthma (CA), past asthma (PA) and chronic bronchitis (CB). The data from GEIRD survey (www.geird.org) have been used in this study. The hierarchic outcome variable was built to achieve mutually exclusive diseases, i.e. Controls (no respiratory disorders, n=549), CA (no PA, n=404), PA (no CA, n=185), CB (no CA, PA, n=92). Multinomial logistic regressions were applied to analyze associations, adjusting for age, BMI, sex, cohort, centre, smoke, comorbidities and alcohol. Relative Risk Ratios (RRR) for one standard deviation increase were adduced for all biomarkers. Glutathione (mg/ml) was higher in subjects with CB (RRR=1.77, CI(1.18-3.07)). FeNO (ppm) was higher in CA (RRR=1.47, CI(1.19-1.82)). Basophils (e+06/ml) had higher levels in CA (RRR=1.48, CI(1.20-1.84)) and CB(RRR=1.51, CI(1.01-2.25)); eosinophils (e+06/ml) were higher in CA (RRR=2.37, CI(1.79-3.13)), PA (RRR=1.79, CI(1.30-2.47)) and CB (RRR=2.14, CI(1.42-3.22)); leucocytes (e+06/ml) were increased in CA (RRR=1.34, CI(1.07-1.67)); lymphocytes (e+06/ml) had higher levels in CA (RRR=1.27, CI(1.03-1.55)) and CB (RRR=1.53, CI(1.05-2.25)). Our results showed that biomarkers of inflammation and OS were differently associated with asthma and chronic bronchitis, suggesting that they might be useful in phenotyping respiratory diseases

Increased susceptibility of LDL to in vitro oxidation in patients with insulin-dependent and non-insulin-dependent diabetes mellitus

The susceptibility of LDL to copper-catalyzed oxidation was evaluated in 24 patients with insulin-dependent and 16 patients with non-insulin-dependent diabetes mellitus, 14 abdominal and 14 gluteal-femoral obese women, 22 familial hypertriglyceridemic and 28 control subjects. Differences in the LDL susceptibilities were studied by measuring the changes of fluorescence intensity and expressed as lag-phase. The lag-phase was significantly shorter in patients with insulin-dependent, non-insulin-dependent diabetes mellitus, abdominal obesity and familial hypertriglyceridemic patients than in gluteal-femoral obese subjects and controls (p < 0.01). The shortest lag-phase was found in familial hypertriglyceridemic patients while intermediate values were found in insulin-dependent, non-insulin-dependent and abdominal obese patients who had only a slight increase in triglyceride values. Similarly the lowest value of the LDL cholesterol to protein ratio, as expression of LDL particle size, was found in familial hypertriglyceridemic patients (p < 0.01), while the patients with insulin-dependent, non-insulin-dependent diabetes mellitus and abdominal obesity had intermediate values. The ratio was found to be directly correlated with the length of the lag-phase (r = 0.87, p < 0.001). In spite of similar triglyceride and cholesterol to protein ratio values, however, the length of the lag-phase was significantly shorter in patients with insulin-dependent diabetes mellitus than in those with abdominal obesity. So it is concluded that the different susceptibility to oxidation found in the different groups of patients is only partially explained by plasma triglyceride values

The susceptibility of LDL to in vitro oxidation is increased in hypercholesterolemic patients and its relation to preformed lipid hydroperoxides in native LDL

It has been suggested that the oxidative modification of low-density lipoprotein (LDL) plays a major role in atherogenesis. We evaluated the oxidative resistance to copper-induced oxidative changes of LDL derived from patients affected by type IIa hyperlipoproteinemia compared with healthy subjects and faced the question of the importance of the antioxidants and polyunsaturated fatty acids (PUFAs) contained in LDL in determining its variability. LDL isolated from the plasmas of 25 subjects affected by familial hypercholesterolemia and 15 control subjects was oxidatively modified with Cu2+ in vitro, and the differences in LDL susceptibilities (lag and propagation phases) to lipid peroxidation were studied by measuring the changes in fluorescence intensity. LDL alpha-tocopherol and PUFAs were also measured. The lag phase was significantly lower and the propagation phase significantly higher in the type IIa patients than in control subjects (p < 0.01). The linoleic and arachidonic acids, expressed as percentage of total LDL fatty acids, were significantly higher in type IIa patients than in the control subjects (p < 0.01). There was a positive significant correlation between the LDL cholesterol and the linoleic and arachidonic acids as percentage of total LDL fatty acids (p < 0.01). Both linoleic and arachidonic acids turned out to be negatively correlated with the lag phase and positively with the propagation phase (p < 0.01). The concentration of LDL alpha-tocopherol was similar in the two groups. Therefore, type IIa patients have a greater susceptibility to LDL oxidation than control subjects. This may be due to a relative higher concentration of linoleic and arachidonic acids in LDL derived from patients with familial hypercholesterolemia

Biomarkers related to respiratory symptoms and lung function in adults with asthma

Background: There is a need for easily measurable biomarkers that are able to identify different levels of asthma severity. Aim: To assess the association between peripheral blood cell counts, fractional nitric oxide in exhaled air (FeNO), urinary biomarkers of oxidative stress (8-hydroxy-2'-deoxyguanosine and 8-isoprostane), and asthma severity in adult patients from the general population. Methods: In the Gene Environment Interactions in Respiratory Diseases study, 287 subjects with asthma (aged 20\u201364) were identified from the general population in Verona (Italy) (2008\u20132010). Self-reported asthma attacks, asthma-like symptoms and the use of hospital services in the past year were synthesized in a score of respiratory symptoms (SRS). The association of biomarkers with SRS and lung function measures (pre-bronchodilator FEV1% predicted and FEV1/FVC) was assessed using quasi-Poisson and Gaussian regression models, respectively. Results: Eosinophils (ratio of expected scores: RES[95%CI] = 1.19[1.09,1.30]), basophils (RES[95%CI] = 1.24[1.10,1.40]), lymphocytes (RES[95%CI] = 1.27[1.12,1.45]) and FeNO (RES[95%CI] = 1.18[1.02,1.37]) were positively associated with SRS. However, only eosinophils (RES[95%CI] = 1.15[1.02,1.30]) and lymphocytes (RES[95%CI] = 1.25[1.06,1.47]) showed an independent association. Furthermore, eosinophils (change in the expected outcome for 1-SD increase: CEO[95%CI] = 121.18[ 122.09, 120.27]%), basophils (CEO[95%CI] = 121.24[ 122.16, 120.33]%) and lymphocytes (CEO[95%CI] = 121.07[ 121.99, 120.14]%) were individually, but not independently, associated with FEV1/FVC. Finally, neutrophils were negatively associated with FEV1% predicted (CEO[95%CI] = 122.22[ 124.00, 120.44]%). Conclusions: We identified a pattern of association between a set of biomarkers and asthma endotypes in adult patients from the general population, which could improve understanding of the heterogeneity and severity of the disease and could be useful in defining targeted therapeutic approaches