Correction to: Oxidative stress and Nrf2 expression in peripheral blood mononuclear cells derived from COPD patients: an observational longitudinal study.

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Oxidative stress and Nrf2 expression in peripheral blood mononuclear cells derived from COPD patients: an observational longitudinal study.

BACKGROUND: A persistent low inflammatory-oxidative status and the inadequacy of the antioxidant nuclear factor-E2-related factor 2 (Nrf2) have been implicated in chronic obstructive pulmonary disease (COPD) progression. Therefore this study was aimed to assess the association between lung function decline and oxidative-inflammatory markers and Nrf2 signaling pathway expression in peripheral blood mononuclear cells (PBMCs) over time. METHODS: 33 mild-moderate COPD outpatients (mean age 66.9 ± 6.9 years) were age-sex matched with 37 no-COPD subjects. A clinical evaluation, blood sampling tests and a spirometry were performed at baseline and after a mean follow-up of 49.7 ± 6.9 months. RESULTS: In COPD, compared to no-COPD, we found a faster lung function decline at follow-up. Although similar prevalence of smoking, hypertension, diabetes and dyslipidemia, systemic markers of inflammation (hs-CRP and white blood cells, WBCs) and oxidative stress (8-isoprostane) were significantly increased in COPD at follow-up, while the antioxidant glutathione (GSH) was significantly reduced. Moreover the expression of Nrf2 and of Nrf2-related genes heme oxygenase (HO)-1 and glutamate-cysteine ligase catalytic (GCLC) subunit in PBMCS were significantly down-regulated in COPD at follow-up, whereas no changes were observed in no-COPD. The percent variation (Δ) of FEV1 detected after the follow-up in COPD patients was directly correlated with ΔNrf2 (r = 0.826 p < 0.001), ΔHO-1 (r = 0.820, p < 0.001) and ΔGCLC (r = 0.840, p < 0.001). Moreover ΔFEV1 was also directly correlated with ΔGSH (r = 0.595, p < 0.01) and inversely correlated with Δ8-iso (r = - 0.587, p < 0.01) and with baseline smoking history (r = - 0.39, p < 0.03). No correlation was found between ΔFEV1, ΔCRP and ΔWBCs. By means of hierarchical stepwise multiple linear regression, taking into account other baseline key factors related to FEV1, ΔNrf2, ΔHO-1and ΔGCLC were found to be significant predictors of ΔFEV1, explaining 89.5% of its variance. CONCLUSIONS: Although our results must be confirmed in larger trial they suggest that the down-regulation of Nrf2/ARE gene expression in PBMCs may be one of the determinants of FEV1 decline and of COPD progression. Therefore the future possibility to counteract Nrf2 decline in COPD patients may help in reducing the negative effects of the oxidative stress-induced progression of the disease.

Enhanced levels of oxidized low-density lipoprotein prime monocytes to cytokine overproduction via upregulation of CD14 and toll-like receptor 4 in unstable angina.

OBJECTIVES: The purpose of this study was to establish whether oxidized low-density lipoprotein (oxLDL) contributes to cytokine overproduction via upregulation of CD14 and toll-like receptor-4 (TLR-4) expression on circulating monocytes of unstable angina (UA) patients. METHODS AND RESULTS: Expression of CD14 and TLR-4 on circulating monocytes, and the concentration of plasma oxLDL, (interleukin [IL])-6, IL-1 beta, IL-8, tumor necrosis factor (TNF)-alpha, monocyte chemoattractant protein-1 (MCP-1) were measured in 27 control (C) subjects, 29 patients with stable angina (SA), and 27 with UA. CD14 and TLR-4 expression on monocytes and circulating IL-6, IL-1 beta, and oxLDL were higher in UA than in SA and C subjects (P<0.001). In in vitro experiments, oxLDL increased CD14 and TLR-4 expression (P<0.001) in control monocytes as well as IL-6, IL-1 beta, and at a lower extent TNF-alpha and MCP-1 levels in the supernatant (P from <0.05 to <0.001). The preincubation of sera derived from UA patients but with control monocytes also induced a significant increase of CD14 and TLR-4 expression (P<0.001) and of IL-6 and IL-1 beta production (P<0.001) in the supernatant. CONCLUSIONS: In UA patients oxLDL may contribute to monocyte overproduction of some cytokines by upregulating CD14 and TLR-4 expression.

Determination of lipid hydroperoxides in native low-density lipoprotein by a chemiluminescent flow-injection assay.

Lipid hydroperoxides have been implicated in the pathogenesis of atherosclerosis. This work was therefore set up to obtain a fast and specific chemiluminescent assay for measuring hydroperoxides in native low-density lipoprotein (LDL). The apparatus was a complete HPLC system including two pumps, an autosampler, a computer and a chemiluminescent detector with a T-mixing coil in the place of the column. Samples were injected from the autosampler and mixed with luminescent reagent (3 microM luminol and 1 microM microperoxidase in 0.1 M carbonate buffer (pH 10)) in the T-piece. To generate a calibration curve, linoleic acid hydroperoxide was obtained by incubating soybean lipoxygenase with linoleic acid. The calculated conjugated diene concentration was in good agreement with the nominal linoleic acid hydroperoxide concentration. The chemiluminescence was linear with the amount of linoleic acid hydroperoxide injected and the detection limit was about 3 pmol linoleic acid hydroperoxide. The chemiluminescence induced by copper-oxidized LDL was linear with concentration; the detection limit, when compared with linoleic acid hydroperoxide, was similar. The reproducibility of the linoleic acid hydroperoxide and of oxidized LDL hydroperoxide was examined in single pools. The coefficient of variation on the triplicates of each pool was about 3%. The titre of the linoleic acid hydroperoxide and oxidized LDL peroxides was quite stable for at least 10 days when stored under argon at 4 degrees C in the presence of EDTA. The mean value of the LDL hydroperoxides in 16 control subjects was 145.20 +/- 98.81 pmol/mg LDL protein. In conclusion, the microperoxidase-luminol-dependent chemiluminescence flow-injection assay is a rapid, sensitive and selective method for measuring lipid hydroperoxides in native LDL.

Troglitazone reduces LDL oxidation and lowers plasma E-selectin concentration in NIDDM patients.

Troglitazone, an oral antidiabetic agent with antioxidant properties, has previously been shown to increase the resistance of LDL to oxidation in vitro and in vivo in healthy volunteers. In a randomized, placebo-controlled, parallel-group study in 29 patients with NIDDM, we tested the effect of troglitazone (200 mg once daily) on the resistance of LDL to oxidation and on circulating levels of preformed lipid hydroperoxides and the adhesion molecule E-selectin. Resistance of LDL to oxidation was assessed by measuring 1) fluorescence development induced by copper treatment (lag phase), and 2) amount of thiobarbituric acid-reactive substances (TBARS) generated by incubation with umbilical vein endothelial cells. At 8 weeks, the lag phase was increased by 23% (P < 0.01 by analysis of covariance [ANCOVA]) in the patients receiving troglitazone (n = 18) compared with the group receiving placebo (n = 11). At the same time, TBARS were 3.63 +/- 0.10 nmol/l (vs. 5.32 +/- 0.10 nmol/l in the placebo group, P = 0.009), LDL hydroperoxide concentration was reduced from 1.48 +/- 0.03 to 1.19 +/- 0.03 ng/mg (no change in the placebo group, P < 0.01), and plasma E-selectin levels decreased from 56.5 +/- 2.33 to 43.7 +/- 1.77 microg/l (no change in the placebo group, P < 0.01). In NIDDM, troglitazone may slow down the development of atherosclerosis by modifying LDL-related atherogenic events.

Endoplasmic reticulum stress and Nrf2 repression in circulating cells of type 2 diabetic patients without the recommended glycemic goals.

Endoplasmic reticulum (ER) stress plays a role in the pathogenesis of type 2 diabetes mellitus (T2DM), with activation of the unfolded protein response (UPR) and ER apoptosis in ß-cells. The aim of the study is investigating the role of the prolonged glycemic, inflammatory, and oxidative impairment as possible UPR and ER apoptosis inductors in triggering the ER stress response and the protective nuclear erythroid-related factor 2 (Nrf2)/antioxidant-related element (ARE) activation in peripheral blood mononuclear cells (PBMC) of T2DM patients without glycemic target. Oxidative stress markers (oxidation product of phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine [oxPAPC], and malondialdehyde [MDA]), the UPR and ER apoptosis, the activation of the pro-inflammatory nuclear factor-kappa B (NF-kB) with its inhibitory protein inhibitor-kBα, and the expression of the protective Nrf2 and heme oxygenase-1 (HO-1) were evaluated in PBMC of 15 T2DM patients and 15 healthy controls (C). OxPAPC concentrations (in PBMC and plasma), MDA levels (in plasma), the expressions of the glucose-regulated protein 78 kDa (or BiP) as representative of UPR, and of the CCAAT/enhancer-binding protein homologous protein as representative of ER apoptosis were significantly higher (p < 0.01) in T2DM with respect to C. IkBα expression was significantly lower (p < 0.01) in T2DM as well as Nrf2 and HO-1. In vitro experiments demonstrated that hyperglycemic conditions, if prolonged, were NF-kB inductors, without a corresponding Nrf2/ARE response. In PBMC of T2DM without glycemic target achievement, there is an activation of the UPR and of the ER apoptosis, which may be related to the chronic exposure to hyperglycemia, to the augmented inflammation, and to the augmented oxidative stress, without a corresponding Nrf2/ARE defense activation.

Antioxidants inhibit the expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 induced by oxidized LDL on human umbilical vein endothelial cells.

The oxidative modification of low density lipoprotein (LDL) and the endothelial expression of adhesion molecules are key events in the pathogenesis of atherosclerosis. In this study we evaluated the effect of oxidized LDL on the expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on human umbilical vein endothelial cells (HUVECs). The hypothesis that oxidized LDL functions as a prooxidant signal was also evaluated, by studying the effect of different radical-scavenging antioxidants on expression of adhesion molecules. LDL was oxidized by using Cu2+, HUVECs or phospholipase A2 (PLA2)/ soybean lipoxygenase (SLO), the degree of oxidation being measured as thiobarbituric acid-reactive substances (TBARS) and conjugated dienes (CD). Exposure of 200 micrograms/ml of native LDL to 1 microns Cu2+, HUVECs and to PLA2/ SLO resulted in four- to fivefold higher levels of TBARS and CD than in native LDL. Cu(2+)-(1 microM), HUVEC-, and PLA2/SLO-oxidized LDL caused a dose-dependent, significant increase of ICAM-1 and VCAM-1 (p < .01). The expression of E-selectin did not change. LDL oxidized with a 2.5 and 5 microM Cu2+ did not increase ICAM-1 and VCAM-1 significantly. Both the Cu(2+)- and HUVEC-oxidized LDL, subjected to dialysis and ultrafiltration, induced ICAM-1 and VCAM-1 expression. After incubation with the ultrafiltrate, the expression of ICAM-1 and VCAM-1 was not significantly different from that obtained with native LDL. LDL pretreated with different antioxidants (vitamin E and probucol) and subjected to oxidation by Cu2+ and HUVECs induced a significantly lower expression of ICAM-1 and VCAM-1 than nonloaded LDL (p < .01). The pretreatment of HUVECs with vitamin E and probucol significantly reduced the expression of VCAM-1 on HUVECs induced by oxidized LDL (p < .01); the effect on ICAM-1 was much less evident. In conclusion, oxidized LDL can induce the expression of different adhesion molecules on HUVECs; this induction can be prevented by pretreating either the LDL or the cells with radical-scavenging antioxidant.

Effect of plasma on the degradation of hydroperoxides of unesterified linoleic acid and copper-peroxidized LDL.

The determination of lipid hydroperoxides in plasma and lipoproteins recently reached a clinical relevance in disorders such as atherosclerosis, where oxidative reactions have been suggested to play a fundamental pathogenetic role. The peroxide content of lipoproteins is usually measured after ultracentrifugation and extraction. During this procedure, some peroxides might decompose causing a too low recovery. To screen this possibility, the disappearance, in the presence of human plasma, of hydroperoxides of linoleic acid and Cu-oxidized low density lipoprotein (LDL) have been investigated, using both a iodometric titration and an enzymatic assay. While only in the presence of GSH plasma decomposes linoleic acid hydroperoxides quite rapidly, peroxides in Cu-oxidized LDL were stable both in presence as well as in absence of GSH. This indicated that lipid hydroperoxides are stable in plasma and that peroxides of Cu-oxidized LDL are not substrate for the glutathione-dependent peroxidase activity in plasma. The relevant decrease of the iodometric titre of LDL peroxides observed in the presence of elevated amounts of plasma was shown to be artifactual, since some compounds extracted from plasma do react with iodine generated by peroxides. Whole plasma itself, indeed, has been shown to reduce back to I- appreciable amount of free iodine.

Effects of guar gum on plasma lipoproteins and apolipoproteins C-II and C-III in patients affected by familial combined hyperlipoproteinemia.

In recent years, guar gum has been shown to be a potent hypocholesterolemic agent. The effects of this fiber on triglycerides are less clear. In order to evaluate the influence of guar supplementation on plasma lipoproteins and apolipoprotein C-II (apoC-II) and apolipoprotein C-III (apoC-III) isoforms (apoC-III2, apoC-III1, apoC-III0), 16 g/day of guar gum were administered to 12 outpatients affected by familial combined hyperlipoproteinemia for a period of 60 days. Mean total cholesterol and triglyceride levels significantly decreased after 15 days of treatment and persisted reduced at the 30th and 60th day of guar supplementation. While low-density lipoprotein cholesterol paralleled the reduction of total plasma cholesterol, we did not observe any change in the cholesterol content of high-density lipoprotein (HDL) subfractions (HDL2 and HDL3) during the study. A redistribution of the relative content of very low-density lipoprotein apoC-III isoforms with a significant increase of apoC-III1 and a decrease of apoC-III0 was observed after 15, 30, and 60 days of guar gum administration. The results show that guar gum reduces not only cholesterol but also triglyceride levels in patients affected by familial combined hyperlipoproteinemia. Further studies are needed to confirm the suggestion that the different distribution of very low-density lipoprotein apoC-III isoforms induced by guar supplementation may influence the behavior of plasma triglycerides.

Long-term effect of a low-fat, high-carbohydrate diet on plasma lipids of patients affected by familial endogenous hypertriglyceridemia.

We evaluated the effect of a low-fat, high carbohydrate (LFHC) diet on plasma lipids in 10 patients affected by familial endogenous hypertriglyceridemia. All the patients studied underwent a base-line period of 4 wk, a 12-wk intervention period, and an 8-wk switch-back period. During the control periods patients consumed approximately 45% of energy as fat and approximately 40% as carbohydrate. During the intervention period they consumed an isocaloric diet containing approximately 25% of energy as fat and approximately 60% as carbohydrate. Total plasma triglyceride and cholesterol levels decreased significantly after 45 and 90 d of treatment (p less than 0.01). The reduction of plasma cholesterol was associated mostly with the decrease in VLDL cholesterol (p less than 0.01) while LDL cholesterol increased at days 45 and 90 of the LFHC diet (p less than 0.01). Finally, we observed a significant increase in HDL cholesterol both at days 45 and 90 of the LFHC diet (p less than 0.01). The LFHC diet we used may be an useful tool in the management of hypertriglyceridemia.

Predisposition to LDL oxidation in patients with and without angiographically established coronary artery disease.

Oxidative modification of low density lipoprotein (LDL) may play an important role in the mechanism of atherosclerotic damage to blood vessels. In the present study the LDL isolated from the plasmas of 73 coronary artery disease (CAD) patients, 28 valvular heart disease (VHD) patients, 59 subjects affected by type IIa hyperlipoproteinemia and 71 controls was oxidatively modified by incubation with copper ions. In 15 CAD and 15 Type IIa patients and 15 controls the LDL chemical composition and polyunsaturated fatty acid (PUFA) content were also measured. Differences in the LDL susceptibilities to lipid peroxidation were studied by measuring the changes of fluorescence intensity. The lag phase in the CAD patients was found to be significantly lower than in the VHD and controls (P < 0.001). The lag phase in the type IIa patients was significantly higher than in the CAD patients (P < 0.01), and significantly lower than the VHD and controls (P < 0.01). The LDL isolated from the type IIa patients had an increase in the relative content of free and esterified cholesterol (P < 0.05), while the CAD patients had a decrease in the relative content of free cholesterol (P < 0.05), and an increase in the relative content of protein (P < 0.05). The lowest value of the LDL cholesterol to protein ratio and LDL size, was found in the CAD patients (P < 0.05). When expressed in micrograms/mg LDL cholesterol, the concentration of the LDL PUFAs was significantly higher in the CAD group than in the others (P < 0.05). The LDL alpha-tocopherol concentration was quite similar in the different groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Lacidipine inhibits the activation of the transcription factor NF-kappaB and the expression of adhesion molecules induced by pro-oxidant signals on endothelial cells.

OBJECTIVE: The adhesion of monocytes to endothelium, an early event in atherosclerosis is mediated by cell adhesion molecules. Signal-transduction pathways for these binding molecules include the translocation of the transcription factor NF-kappaB; moreover, intracellularly generated oxygen-derived free radicals play a major role in this process. In this study we evaluated the extent to which lacidipine, a calcium antagonist with antioxidant properties, affects the expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin on human umbilical vein endothelial cells, induced by different pro-oxidant signals such as oxidized low density lipoprotein (LDL) and tumor necrosis factor-alpha (TNF-alpha). METHODS: We incubated 5 micromol/l Cu2+-oxidized LDL and TNF-alpha (2 ng/ml) with human umbilical vein endothelial cells for 48 and 6 h, respectively. ICAM-1, VCAM-1 and E-selectin were measured by flow cytometry. NF-kappaB was evaluated by electrophoretic mobility shift assay. RESULTS: The incubation of 5 micromol/l Cu2+-oxidized LDL not only caused a dose-dependent increase in ICAM-1, VCAM-1 and E-selectin (P < 0.001), but also synergically increased their TNF-alpha-induced expression (P < 0.001). The addition of lacidipine to human umbilical vein endothelial cells significantly reduced the expression of ICAM-1, VCAM-1 and E-selectin induced by TNF-alpha alone or with oxidized LDL (P < 0.001). The reduction in adhesion molecule expression caused by lacidipine was paralleled by a significant fall in NF-kappaB translocation. CONCLUSIONS: The results suggest that lacidipine may have prevented NF-kappaB-mediated adhesion molecule expression by exerting its effects on oxygen-derived free radicals. The results support previous observations that lacidipine may have therapeutic effects in atherosclerosis.

Oxidized low-density lipoprotein increases the production of intracellular reactive oxygen species in endothelial cells: inhibitory effect of lacidipine.

OBJECTIVE: The mechanisms by which oxidized low-density lipoprotein (ox-LDL) induces the expression of adhesion molecules on endothelial cells (HUVECs) are still not clear. The signal transduction pathways for these binding molecules include the translocation of the transcription factor NF-kB and the intracellular reactive oxygen species (ROS) are said to play a key role in this process. Aim of this study was (1) to evaluate the effect of ox-LDL on intracellular production of ROS in culture of HUVECs; (2) to evaluate if the intracellular increase of ROS induced by ox-LDL is mediated by the binding to a specific endothelial receptor; (3) to ascertain if lacidipine can decrease ox-LDL-induced ROS production in HUVECs. METHODS: Five microM Cu2+ ox-LDL were incubated with HUVECs for 5 min. 2',7'-Dichlorofluorescein (DCF) as an expression of intracellular ROS production, was measured by flow cytometry. RESULTS: ox-LDL induced a significant dose-dependent increase in DCF production (P < 0.001) through the binding to a specific receptor. The preincubation of HUVECs with radical scavengers compounds and lacidipine significantly reduced (P < 0.001) the ox-LDL-induced DCF production. CONCLUSIONS: ox-LDL increased the intracellular formation of ROS through the ligation to a specific endothelial receptor. Preincubation of HUVECs with lacidipine, a calcium antagonist with antioxidant properties, significantly reduced the intracellular ROS formation induced by ox-LDL. We propose that the effect of lacidipine on adhesion molecule expression and on NF-kB activation can be explained by its effect on intracellular ROS formation.

Effect of calcium antagonist or beta blockade treatment on nitric oxide-dependent vasodilation and oxidative stress in essential hypertensive patients.

OBJECTIVES: Essential hypertension is associated with impaired endothelium-dependent vasodilation caused by oxygen free radical-induced nitric oxide (NO) breakdown. Since calcium antagonists can improve endothelial function in hypertensive patients, we tested whether this beneficial effect could be related to restoration of NO availability by antioxidant activity. METHODS: In 10 healthy subjects and 20 essential hypertensive patients, we studied forearm blood flow (strain-gauge plethysmography) modifications induced by intrabrachial acetylcholine (from 0.15-15 microg/100 ml per min), bradykinin (0.005-0.05 microg/100 ml per min), two endothelium-dependent vasodilators, and sodium nitroprusside (1-4 microg/100 ml forearm tissue per min), an endothelium independent vasodilator, in the absence and presence of NG-monomethyl-L-arginine (L-NMMA) (100 microg/100 ml forearm tissue per min), an NO synthase inhibitor. RESULTS: In controls, vasodilation to acetylcholine and bradykinin was inhibited by L-NMMA. In hypertensive patients, vasodilation to acetylcholine and bradykinin, but not to sodium nitroprusside, was blunted and resistant to L-NMMA. Hypertensive patients were randomized to a 12-week treatment with lacidipine (4-6 mg/daily) or atenolol (50-100 mg/daily) (n = 10 each group). Lacidipine but not atenolol increased the vasodilation to acetylcholine and bradykinin and restored the inhibiting effect of L-NMMA on endothelium-dependent vasodilation, without affecting the response to sodium nitroprusside. Moreover, lacidipine reduced circulating markers of oxidative stress including plasma and low-density lipoprotein (LDL) hydroperoxides, the susceptibility of LDL to Cu2+-induced oxidation and the reactive oxygen species generated from human umbilical vein endothelial cells after incubation with LDL derived from plasma of the patients. CONCLUSIONS: Lacidipine increases endothelium-dependent vasodilation by restoring NO availability, and this effect possibly is related to antioxidant activity.

Comparative effects of different dihydropyridines on the expression of adhesion molecules induced by TNF-alpha on endothelial cells.

OBJECTIVE: Lacidipine has already been demonstrated to reduce the expression of some adhesion molecules induced by pro-oxidant signals on endothelial cells. In order to verify if this effect is a peculiarity of this molecule, or belongs to other dihydropyridinic compounds (DHPs), the activity of lacidipine was compared with that of lercanidipine, amlodipine, nimodipine and nifedipine. DESIGN AND METHODS: The compounds were incorporated in human umbilical vein endothelial cells (HUVECs) using native low-density lipoprotein as a carrier. The drug concentrations in HUVECs were measured by mass spectrometry. Human recombinant tumour necrosis factor-alpha was then incubated with HUVECs for 7 h at 37 degrees C for adhesion molecule expression. RESULTS: The cellular amount of lacidipine, lercanidipine and amlodipine was similar, while nimodipine and nifedipine were almost undetectable or undetectable, respectively. Lacidipine, at any concentration, determined a dose-dependent significant decrease of the expression of intercellular adhesion molecule-1 (ICAM-1) ICAM-1, vascular cell adhesion molecule-1 (VCAM-1) VCAM-1 and E-selectin (P < 0.01). Lercanidipine and amlodipine determined variable decreases of adhesion molecules at the intermediate and highest concentrations. Nimodipine and nifedipine determined no effect on ICAM-1, VCAM-1 and E-selectin. The lowest IC50, i.e. the concentration determining the 50% reduction of ICAM-1, VCAM-1 and E-selectin expression was obtained with lacidipine for all the adhesion molecules considered (P < 0.01). CONCLUSIONS: It is concluded that the effect of the DHPs used in this study on adhesion molecule expression is determined first by their lipophilicity and then by their intrinsic antioxidant activity.

Oxidized LDL and reduction of the antiaggregating activity of nitric oxide derived from endothelial cells.

Oxidized LDL has been observed to induce abnormalities in endothelial function which may be relevant for the progression of atherosclerotic lesions. We studied in vitro the possible effects of oxidized LDL on the antiaggregating activity of endothelial cells, which is dependent on release of prostacyclin and nitric oxide. We used an experimental model in which cultured human endothelial cells were placed in the aggregometer in contact with human platelets, after blockade of cyclo-oxygenase by adding acetylsalicylic acid. In this way the antiaggregant effect of endothelial cells was dependent on the release of nitric oxide alone; prevention of antiaggregant activity by preincubation of endothelial cells with 300 microM L-NG-mono-methylarginine confirmed this. When this system was used, endothelial cells (2-7.5 x 10(5)/ml) almost completely inhibited thrombin-induced (0.02-0.08 U/ml) platelet aggregation (2 x 10(8) platelets/ml), measured according to Born (11.1% +/- 8.5 vs 68.6% +/- 12.6, M +/- SD). This antiaggregating activity was reduced when slightly oxidized LDL 100 micrograms/ml (35.2% +/- 14.9, p < 0.001), but not native LDL 100 micrograms/ml (7.5% +/- 7.6), was added immediately before aggregation was induced. Incubation of endothelial cells with oxidized LDL 100 micrograms/ml for 1 h did not affect the antiaggregating capacity, unless oxidized LDL was present during aggregation (18.3% +/- 10.2 vs 35.8% +/- 9.6, p < 0.02). No significant direct effect of either oxidized or native LDL on stimulated platelet aggregation was observed. Our results indicate that slightly oxidized LDL can reduce the antiaggregating properties of the endothelium, probably by interaction with NO rather than through inhibition of its synthesis.

Can voltammetry measure nitrogen monoxide (NO) and/or nitrites?

Recently, voltammetry with carbon fibre electrodes (CFE) has been implemented for real time measurement of nitrogen monoxide (NO) indicating that it is oxidised at the potential value of nitrites, approximately +700 mV. In contrast, here we show that modified CFE can monitor NO at oxidation potentials different than that of nitrites, i.e. +550 mV. Indeed, at +550 mV a significant increase of amperometric current levels was obtained when NO but not nitrites, were added to a phosphate buffer saline solution (PBS). Differential pulse voltammetry (DPV) supports these findings as two oxidation peaks were obtained when examining air preserved NO; peak 1 at +550 mV and peak 2 at +700 mV, respectively. In contrast, only peak 2 was monitored when nitrites or a solution of NO oxidised in air was added to PBS. Biological support to these in vitro data comes from the observation that the relaxation of an adrenaline-contracted aortic ring produced via addition of NO is concomitant with peak 1 at +550 mV. The relaxation is almost completed before the appearance of peak 2 at +700 mV. Furthermore, in vivo experiments performed in the striatum of rats show that the amperometric signal monitored at +550 mV is responsive to glutamatergic stimulation or inhibition of NO synthase.

Predisposition to LDL oxidation during copper-catalyzed oxidative modification and its relation to alpha-tocopherol content in humans.

The predisposition to LDL oxidation during copper-catalyzed oxidative modification and its relationship with LDL alpha-tocopherol concentration was studied in 41 control subjects. The results show that the predisposition of LDL to oxidation expressed as duration of the inhibition period and rate of the propagation period varied greatly in the controls, but did not correlate with the values of LDL alpha-tocopherol. On the contrary the experiments with alpha-tocopherol incorporated in LDL demonstrate that even small increases of incorporated alpha-tocopherol, under circumstances where other variables were probably largely unaffected, increased proportionally the length of the inhibition period and reduced the rate of the propagation period. The values of LDL alpha-tocopherol achieved after the enrichment turned out to be positively correlated with the duration of the inhibition period and negatively with the rate of the propagation period. Finally the results of this study also show that there was a variability in the LDL alpha-tocopherol decay of different subjects under the same oxidative stress. In our conditions however, the time in which alpha-tocopherol contributed to the LDL protection was much shorter than the mean length of the inhibition period. The results demonstrate that the variability in the predisposition to LDL oxidation during copper-catalyzed oxidative modification is not determined only by the concentration of alpha-tocopherol in LDL and that therefore its value as a sole indicator of antioxidant status is probably inadequate.

The susceptibility of low-density lipoprotein to in vitro oxidation is increased in hypercholesterolemic patients.

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.

Increased susceptibility of LDL to in vitro oxidation in patients on maintenance hemodialysis: effects of fish oil and vitamin E administration.

Oxidized low-density lipoproteins (LDL) play an important role in the pathogenesis of atherosclerosis. An increased sensitivity of red blood cell membranes to lipid peroxidation has been previously demonstrated in patients with chronic renal failure, suggesting that the antioxidant defence of lipoproteins might be impaired. Fish oil supplementation has been proposed in dialysis patients, but it is still unclear if the positive effects of fish oil depend only on its polyunsaturated fatty acid content or on other factors, such as the usually added antioxidants. Moreover, the increased concentration of highly peroxidable n-3 polyunsaturated fatty acids induced by fish oil in LDL particles could favour LDL oxidation and possibly the development of atherosclerosis. The present study was designed to evaluate the susceptibility of LDL to in vitro oxidation (lag phase) and the rate of lipid peroxidation (propagation phase) by fluorescence development during copper exposure in 14 hemodialysis patients. A further aim was to compare the effects on lipid metabolism and LDL oxidation of fish oil supplementation (20 ml containing vitamin E 20 IU as antioxidant) for 30 days and of vitamin E administration (50 IU) for another 30 days. The length of the lag phase and vitamin E concentration were significantly reduced (p < 0.01) in hemodialysis patients and increased significantly (p < 0.01) after administration of both fish oil and vitamin E. Fish oil supplementation also reduced plasma lipids significantly (p < 0.01) and increased the propagation phase (p < 0.01). Our results demonstrate that the susceptibility of LDL to oxidation is enhanced in hemodialysis patients, suggesting a possible relationship between excessive LDL peroxidation and accelerated atherosclerosis. The increased susceptibility of LDL to in vitro oxidation can be explained, at least partially, by a reduced LDL vitamin E concentration. Since fish oil increased the lag phase to the same extent as vitamin E supplementation, the positive effect of fish oil could be partly explained by its antioxidant content.