Biomarkers of oxidative stress: an analytical approach

Oxidative stress is implicated in many pathological processes and results from a disruption of the prooxidant/antioxidant balance. This review will focus on noninvasive biomarkers of radical-induced damage in biological fluids and particularly in blood. Special attention will be addressed to new analytical methods for the measurement of radical-mediated alterations in the integrity of lipids, proteins and DNA.     

Effects of prolonged propranolol treatment on left ventricular remodeling and oxidative stress after myocardial infarction in rats

Mechanisms determining the benefit of beta blockade in patients with heart failure remain incompletely understood but are assumed consequent to prevention of deleterious effects of catecholamines. Recent studies have demonstrated that oxidative stress in congestive heart failure may be related to increased catecholamine levels. The aim of this study was to examine effects of long-term treatment with propranolol on progression of left ventricular (LV) dysfunction, remodeling and oxidative stress on an experimental model of chronic heart failure. Six weeks after myocardial infarction by coronary ligation, Wistar rats were randomized to two groups: 10 weeks of therapy with propranolol (50 mg/kg/day in drinking water) and no treatment (infarcted controls). A third group was sham-operated rats without treatment. Animals were anesthetized for hemodynamic measurements, and hearts were then removed for histologic analysis, papillary muscle contractility study, and oxidative stress measurements using thiobarbituric acid reactive substance (TBARS) determination. Control infarcted rats demonstrated significant alterations of hemodynamic parameters and remodeling with increase of heart weight/body weight, of right ventricular lateral wall thickness, of LV circumference, LV septal area/body weight, and LV papillary muscle weight/body weight as compared with sham. In propranolol-treated rats, hypertrophy of the LV septum, papillary muscle, and right ventricle were similar to those of the infarcted control. Myocardial oxidative stress was significantly increased in control infarcted rats compared with sham, and propranolol prevented such oxidative stress increase. Papillary muscle isometric tension parameters were not significantly different among groups. Propranolol treatment prevented isoprenaline-induced spontaneous papillary muscle activity in vitro. Oxidative stress is increased in the rat model of heart failure secondary to coronary ligation. Long-term treatment with propranolol in vivo does not modify the compensatory process of hypertrophy but completely abolishes the oxidative stress increase and reduces the increased cardiac sensitivity to catecholamine-induced arrhythmias observed in this experimental model of heart failure.     

Metformin decreases intracellular production of reactive oxygen species in aortic endothelial cells

Beyond its antidiabetic activity justifying its use in the treatment of the type 2 diabetes, metformin (MET [dimethylguanidine, Glucophage]) has been shown to exhibit antioxidant properties in vitro, which could contribute to limit the deleterious vascular complications of diabetes. We investigated whether MET, at the pharmacological level of 10 -5 mol/L, was able to modulate intracellular production of reactive oxygen species (ROS) both in quiescent bovine aortic endothelial cells (BAECs) and in BAECs stimulated by a short incubation with high levels of glucose (30 mmol/L, 2 hours) or angiotensin II (10 -7 mol/L, 1 hour). Intracellular ROS production was measured by fluorescence of the DCF (2,7-dichlorodihydrofluorescein) probe. Our results showed that MET was able to reduce the intracellular production of ROS in both nonstimulated BAECs (-20%, P < .05) and BAEC stimulated by high levels of glucose or angiotensin II (-28% and -72%, respectively, P < .01). Experiments performed in the presence of the nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase inhibitor apocynin or the respiratory mitochondrial chain inhibitor rotenone indicated that MET exerted its effect partly through an inhibition of the formation of ROS produced mainly by NAD(P)H oxidase and also, to a lesser extent, by the respiratory mitochondrial chain.     

Blood Pressure-Lowering Response to Amlodipine as a Determinant of the Antioxidative Activity of Small, Dense HDL3

Background and Objective

High-density lipoproteins (HDLs) exert multiple antiatherogenic activities including protection of low-density lipoproteins (LDLs) from oxidative stress. Beneficial effects of calcium channel blockers on cardiovascular disease may in part be related to the reduction of oxidative stress, potentially enhancing the antioxidative activity (AOX) of HDLs. This study aimed to assess the effect of 1 month’s treatment with amlodipine on HDL AOX in hypertensive subjects.

Methods

This was a prospective trial of amlodipine 10 mg/day administered for 1 month in primary-care patients with hypertension (n = 28), 46% of whom were obese and 57% of whom displayed the metabolic syndrome. The main outcome measure was HDL AOX, assessed as the capacity of small, dense HDL3c particles to attenuate LDL oxidation induced in vitro by an azo initiator (AAPH).

Results

Mean (±SD) systolic (SBP) and diastolic (DBP) BP were reduced by amlodipine by 22.1mmHg (±13.2) and 10.4mmHg (±7.5), respectively (p<0.001). Body mass index, waist circumference, and plasma levels of triglycerides, cholesterol, and fasting blood glucose did not change significantly. Amlodipine treatment did not modify HDL3c AOX in the whole study population; changes in AOX were, however, positively correlated with SBP (r = 0.37, p = 0.05 for maximal diene concentration; r = 0.34, p = 0.08 for LDL oxidation rate). When the population was divided into two subgroups according to the BP response to amlodipine (change in SBP below or above the median), HDL3c AOX was significantly improved in hyperresponders (BP-lowering response >22/10 mmHg) as compared with hypo-responders (BP-lowering response <22/10mmHg: mean [± SD] change in the LDL oxidation rate in the presence of HDL3c, ?6.8% [± 11.2] vs +1.9% [±5.2], respectively, p = 0.04; maximal diene concentration, ?8.6% [±13.0] vs +1.9% [±8.2], respectively, p<0.05). By contrast, neither plasma concentrations of oxidized LDL, a marker of systemic oxidative stress, nor the chemical composition of HDL3c were modified between the subgroups.

Conclusions

In hypertensive patients, amlodipine treatment enhanced HDL AOX in subjects who had a BP reduction that exceeded the median response. This effect appears to be secondary to the hypotensive effect, rather than to the direct antioxidant properties, of the drug.     

Protective effects of regulatory amino acids on ischemia-reperfusion injury in the isolated perfused rat liver

OBJECTIVE: Some amino acids (AAs) display potent regulatory activities on cell metabolism, including via anti-oxidative defences. The aim of this study was to evaluate the protective effect of these AAs on warm ischaemia-reperfusion (I/R) injury in the isolated perfused rat liver. MATERIAL AND METHODS: Livers from fasted male Sprague-Dawley rats were isolated and perfused without (control group) or with (AP group) a mixture of regulatory AAs (glutamine, histidine, leucine, methionine, proline, phenylalanine, tryptophan and alanine). After 45 min of perfusion, warm ischaemia was induced for 45 min by clamping the portal vein catheter; thereafter, reperfusion was performed for 30 min. RESULTS: TNF-alpha production was significantly lower in the AP group during reperfusion (Control: 39+/-7 versus AP: 16+/-2 pg min-1 g-1, p<0.05), and lactate dehydrogenase (LDH) release decreased significantly during the last 15 min of reperfusion (Control: 0.13+/-0.03 versus AP: 0.04+/-0.02 IU min-1 g-1, p<0.05), despite similar levels of oxidative stress. The addition of regulatory AAs was not associated with variations in portal flow, bile flow, hepatic glucose or urea metabolism. However, significant changes in intrahepatic glutamine (Control: 1.4+/-0.2 versus AP: 2.6+/-0.5 micromol g-1, p < 0.05) together with higher glutamate release in the AP group (Control: 10.2+/-5.4 versus AP: 42.6+/-10.9 nmol min-1 g-1, p < 0.05) indicated modifications in nitrogen metabolism. CONCLUSIONS: Taken together, the lower TNF-alpha production, suggesting decreased inflammatory response, the decrease in LDH release in the AP group, demonstrating a better preservation of liver viability, and the increase in hepatic glutamine indicate that AAs play an important role in the liver's response to I/R.     

Superoxide production and NADPH oxidase expression in human rheumatoid synovial cells: regulation by interleukin-1β and tumour necrosis factor-α

Objectives to evaluate the rheumatoid synovial cell capacity to produce superoxide anion in response to interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α), and to study the NADPH oxidase involvement in this production. Material and Methods Synovial cells obtained from 7 rheumatoid arthritis (RA), 5 osteoarthritic (OA) patients, and dermal fibroblasts, were stimulated (i) with IL-1β and TNF-α, or (ii) with specific oxidase activators and inhibitors, before studying superoxide production; we also studied NADPH oxidase mRNAs and protein expression, and p47-phox phosphorylation. Results Constitutive superoxide production by RA cells was increased in comparison to OA cells and dermal fibroblasts, and was stimulated by PMA and ionomycin. This production was increased after cytokine treatment of RA synovial cells. Cytokine-induced superoxide production by RA cells was inhibited by iodonium diphenyl or apocynin, suggesting the involvement of NADPH oxidase. RT-PCR and western blot analysis revealed the presence of p47-phox, gp91-phox and Nox4 in RA and OA cells, and in dermal fibroblasts. P47-phox phosphorylation was enhanced after cytokine-treatment in RA and OA cells, suggesting a PKC-mediated up-regulation of NADPH oxidase. Conclusions NADPH oxidase is involved in the superoxide release by RA synovial cells, constitutively and after cytokine up-regulation. These cells express two different homologues (gp91-phox and Nox4). Received 2 August 2005; returned for revision 12 January 2006; returned for final revision 22 May 2006; accepted by J. Di Battista 9 June 2006     

A perfusion bioreactor for engineering bone constructs: an in vitro and in vivo study

A perfusion bioreactor, which was designed based on fluidized bed concepts, was validated for the culture of bone constructs of clinically relevant size. For this study, natural coral has been used as three-dimensional scaffolds. This biomaterial is a microporous, biocompatible, osteoconductive, and absorbable scaffold. This perfusion bioreactor provided a stable environment in terms of osmolarity, pH, and, most importantly, oxidative stress. Bone constructs engineered in this system resulted in significantly higher cell proliferation and homogenous cell distribution than those cultured under static conditions. Particularly relevant to the production of bioengineered bone in a clinical setting, custom-made bone constructs (each one with volume up to 30 cm(3)) could be produced using a such perfusion bioreactor. Last, but not least, the bone constructs of clinically relevant volume thus produced were shown to be osteogenic when transplanted subcutaneously in sheep.     

Serum levels of adhesion molecules ICAM-1 and VCAM-1 and tissue inhibitor of metalloproteinases, TIMP-1, are elevated in patients with autoimmune thyroid disorders: Relevance to vascular inflammation

Serum levels of ICAM-1 (Inter Cellular Adhesion Molecule-1), VCAM-1 (Vascular cell Adhesion Molecule-1-I), TIMP-1 (tissue inhibitor of metalloproteinases 1) and MMP-9 (Metalloproteinase 9) are well established markers of inflammation. The physiopathological link between inflammation, atherosclerosis and autoimmunity is well demonstrated. However, serum levels of these biomarkers in patients with autoimmune-mediated dysthyroidism, including their evolution after improvement of the thyroid disorder have not been assessed. So, we evaluated the circulating levels of these markers in autoimmune and in non-autoimmune-mediated dysthyroid patients, and their evolution after treatment of thyroid disease.We conducted a prospective study to evaluate these markers before and after treatment in hyperthyroid patients (n = 33; 28 patients with autoimmune disease), hypothyroid patients (n = 38; 33 patients with autoimmune disease) and euthyroid subjects (n = 33). At baseline, serum levels of ICAM-1, VCAM-1 and TIMP-1 were significantly elevated in patients with hyperthyroidism as compared to euthyroid and hypothyroid patients (respectively p = 0.0005 and p < 0.0001). In multivariate analysis, the differences remained significant for VCAM-1 and TIMP-1. Median levels of ICAM-1, VCAM-1 and TIMP-1 were significantly higher in patients with autoimmune-mediated dysthyroidism compared to euthyroid patients (respectively p < 0.0001 and p = 0.002). In hyperthyroid patients, ICAM-1, VCAM-1 and TIMP-1 concentrations fell significantly after they had become euthyroid (respectively p = 0.0006; p < 0.0001 and p = 0.0009), although VCAM-1 values remained higher than those observed in the control group (p = 0.005).We found that autoimmune-mediated dysthyroidism were associated with increased peripheral blood concentrations of VCAM-1, ICAM-1 and TIMP-1. Whether these biological abnormalities translate into increase intima remodelling and atherosclerosis remains to be studied.