Prevention of retinal capillary basement membrane thickening in diabetic dogs by a non-steroidal anti-inflammatory drug

AIMS/HYPOTHESIS: To investigate the effect of treatment with the non-steroidal anti-inflammatory drug Sulindac on the early vascular pathology of diabetic retinopathy in the dog, and it's effect on recognised biochemical indices of hyperglycaemia-related pathophysiology. METHODS: Experimental diabetes (streptozotocin/alloxan) was induced in 22 male beagle dogs and 12 of the animals were assigned at random to receive oral Sulindac (10 mg/kg daily). Age- and sex-matched control animals were maintained as non-diabetic controls. After 4 years, several morphological parameters were quantified in the retinal microvasculature of each animal group using an established stereological method. Also, the following diabetes-associated biochemical parameters were analysed: accumulation of advanced glycation end products (AGEs), red blood cell polyol levels and antioxidant status. RESULTS: Diabetes increased red blood cell sorbitol levels when compared to non-diabetic controls (p<or =0.05), however, there was no difference in sorbitol levels between the untreated and the treated diabetic animals. No significant differences were found in red blood cell myoinositol levels between the three groups of animals. Pentosidine and other AGEs were increased two- to three-fold in the diabetic animals (p<or =0.001) although treatment with Sulindac did not affect their accumulation in diabetic skin collagen or alter diabetes-induced rises in plasma malondialdehyde. Retinal capillary basement membrane volume was significantly increased in the untreated diabetic dogs compared to non-diabetic controls or Sulindac-treated diabetic animals (p<or =0.0001). CONCLUSION/INTERPRETATION: This study has confirmed the beneficial effect of a non-steroidal anti-inflammatory drug on the early vascular pathology of diabetic retinopathy. However the treatment benefit was not dependent on inhibition of polyol pathway activity, advanced glycation, or oxidative stress.

Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency

Background: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial oxidative stress and hypertrophic remodeling. Up-regulation of the cardiomyocyte adrenomedullin (AM) / intermedin (IMD) receptor signaling cascade is also apparent in NO-deficient cardiomyocytes: augmented expression of AM and receptor activity modifying proteins RAMP2 and RAMP3 is prevented by blood pressure normalization while that of RAMP1 and intermedin (IMD) is not, indicating that the latter is regulated by a pressure-independent mechanism. Aims: to verify the ability of an anti-oxidant intervention to normalize cardiomyocyte oxidant status and to investigate the influence of such an intervention on expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes. Methods: NO synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 35mg/kg/day) was given to rats for 8 weeks, with/without con-current administration of antioxidants (Vitamin C (25mg/kg/day) and Tempol (25mg/kg/day)). Results: In left ventricular cardiomyocytes isolated from L-NAME treated rats, increased oxidative stress was indicated by augmented (3.6 fold) membrane protein oxidation, enhanced expression of catalytic and regulatory subunits of pro-oxidant NADPH oxidases (NOX1, NOX2) and compensatory increases in expression of anti-oxidant glutathione peroxidase and Cu/Zn superoxide dismutases (SOD1, SOD3). Vitamin C plus Tempol did not reduce systolic blood pressure but normalized augmented plasma levels of IMD, but not of AM, and in cardiomyocytes: (i) abolished increased membrane protein oxidation; (ii) normalized augmented expression of prepro-IMD and RAMP1, but not prepro-AM, RAMP2 and RAMP3; (iii) attenuated (by 42%) increased width and normalized expression of hypertrophic markers, skeletal-�-actin and prepro-endothelin-1 similarly to blood pressure normalization but in contrast to blood pressure normalization did not attenuate augmented brain natriuretic peptide (BNP) expression. Conclusion: normalization specifically of augmented IMD/RAMP1 expression in NO-deficient cardiomyocytes by antioxidant intervention in the absence of blood pressure reduction indicates that these genes are likely to be induced directly by myocardial oxidative stress. Although oxidative stress contributed to cardiomyocyte hypertrophy, induction of IMD and RAMP1 is unlikely to be secondary to cardiomyocyte hypertrophy.

A Dual Role for Lecithin:Cholesterol Acyltransferase (EC 2.3.1.43) in Lipoprotein Oxidation

Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme involved in lipoprotein metabolism. It mediates the transesterification of free cholesterol to cholesteryl ester in an apoprotein A-I-dependent process. We have isolated purified LCAT from human plasma using anion-exchange chromatography and characterized the extracted LCAT in terms of its molecular weight, molar absorption coefficient, and enzymatic activity. The participation of LCAT in the oxidation of very low density lipoproteins (VLDL) and low-density lipoproteins (LDL) was examined by supplementing lipoproteins with exogenous LCAT over a range of protein concentrations. LCAT-depleted lipoproteins were also prepared and their oxidation kinetics examined. Our results provide evidence for a dual role for LCAT in lipoprotein oxidation, whereby it acts in a dose-responsive manner as a potent pro-oxidant during VLDL oxidation, but as an antioxidant during LDL oxidation. We believe this novel pro-oxidant effect may be attributable to the LCAT-mediated formation of oxidized cholesteryl ester in VLDL, whereas the antioxidant effect is similar to that of chain-breaking antioxidants. Thus, we have demonstrated that the high-density lipoprotein-associated enzyme LCAT may have a significant role to play in lipoprotein modification and hence atherogenesis. (C) 2007 Elsevier Inc. All rights reserved.

Molecular detection of exercise-induced free radicals following ascorbate prophylaxis in type 1 diabetes mellitus: a randomised controlled trial

Aims/hypothesis: Patients with type 1 diabetes mellitus are more susceptible than healthy individuals to exercise-induced oxidative stress and vascular endothelial dysfunction, which has important implications for the progression of disease. Thus, in the present study, we designed a randomised double-blind, placebo-controlled trial to test the original hypothesis that oral prophylaxis with vitamin C attenuates rest and exercise-induced free radical-mediated lipid peroxidation in type 1 diabetes mellitus. Methods: All data were collected from hospitalised diabetic patients. The electron paramagnetic resonance spectroscopic detection of spin-trapped a-phenyl-tert-butylnitrone (PBN) adducts was combined with the use of supporting markers of lipid peroxidation and non-enzymatic antioxidants to assess exercise-induced oxidative stress in male patients with type 1 diabetes (HbA1c 7.9±1%, n=12) and healthy controls (HbA1c 4.6±0.5%, n=14). Following participant randomisation using numbers in a sealed envelope, venous blood samples were obtained at rest, after a maximal exercise challenge and before and 2 h after oral ingestion of 1 g ascorbate or placebo. Participants and lead investigators were blinded to the administration of either placebo or ascorbate treatments. Primary outcome was the difference in changes in free radicals following ascorbate ingestion. Resuts: Six diabetic patients and seven healthy control participants were randomised to each of the placebo and ascorbate groups. Diabetic patients (n=12) exhibited an elevated concentration of PBN adducts (p<0.05 vs healthy, n=14), which were confirmed as secondary, lipid-derived oxygen-centred alkoxyl (RO•) radicals (a nitrogen=1.37 mT and aßhydrogen=0.18 mT). Lipid hydroperoxides were also selectively elevated and associated with a depression of retinol and lycopene (p<0.05 vs healthy). Vitamin C supplementation increased plasma vitamin C concentration to a similar degree in both groups (p<0.05 vs pre-supplementation) and attenuated the exercise-induced oxidative stress response (p<0.05 vs healthy). There were no selective treatment differences between groups in the primary outcome variable. Conclusions/ interpretation: These findings are the first to suggest that oral vitamin C supplementation provides an effective prophylaxis against exercise-induced free radical-mediated lipid peroxidation in human diabetic blood.

Role of creatine supplementation on exercise-induced cardiovascular function and oxidative stress

Many degenerative diseases are associated with increased oxidative stress. Creatine has the potential to act as an indirect and direct antioxidant; however, limited data exist to evaluate the antioxidant capabdities of creatine supplementation within in vivo human systems. This study aimed to investigate the effects of oral creatine supplementation on markers of oxidative stress and antioxidant defenses following exhaustive cycling exercise. Following preliminary testing and two additional familiarization sessions, 18 active males repeated two exhaustive incremental cycling trials (T1 and T2) separated by exactly 7 days. The subjects were assigned, in a double-blind manner, to receive either 20 g of creatine (Cr) or a placebo (P) for the 5 days preceding T2. Breath-by-breath respiratory data and heart rate were continually recorded throughout the exercise protocol and blood samples were obtained at rest (preexercise), at the end of exercise (postexercise), and the day following exercise (post24 h). Serum hypdroperoxide concentrations were elevated at postexercise by 17 +/- 5% above preexercise values (p = 0.030). However, supplementation did not influence lipid peroxidation (serum hypdroperoxide concentrations), resistance of low density lipoprotein to oxidative stress (t(1/2max) LDL oxidation) and plasma concentrations of non-enzymatic antioxidants (retinol, alpha-carotene, beta-carotene, alpha-tocopherol, gamma-tocopherol, lycopene and vitamin Q. Heart rate and oxygen uptake responses to exercise were not affected by supplementation. These findings suggest that short-term creatine supplementation does not enhance non-enzymatic antioxidant defence or protect against lipid peroxidation induced by exhaustive cycling in healthy males.

Blood levels of vitamin C, carotenoids and retinol are inversely associated with cataract in a north Indian population

PURPOSE. To examine the association of blood antioxidants with cataract.

The AGE inhibitor pyridoxamine inhibits development of retinopathy in experimental diabetes

We examined the ability of pyridoxamine (PM), an inhibitor of formation of advanced glycation end products (AGEs) and lipoxidation end products (ALEs), to protect against diabetes-induced retinal vascular lesions. The effects of PM were compared with the antioxidants vitamin E (VE) and R-alpha-lipoic acid (LA) in streptozotocin-induced diabetic rats. Animals were given either PM (1 g/l drinking water), VE (2,000 IU/kg diet), or LA (0.05%/kg diet). After 29 weeks of diabetes, retinas were examined for pathogenic changes, alterations in extracellular matrix (ECM) gene expression, and accumulation of the immunoreactive AGE/ALE N-epsilon-(carboxymethyl)lysine (CML). Acellular capillaries were increased more than threefold, accompanied by significant upregulation of laminin immunoreactivity in the retinal microvasculature. Diabetes also increased mRNA expression for fibronectin (2-fold), collagen IV (1.6-fold), and laminin beta chain (2.6-fold) in untreated diabetic rats compared with nondiabetic rats. PM treatment protected against capillary drop-out and limited laminin protein upregulation and ECM mRNA expression and the increase in CML in the retinal vasculature. VE and LA failed to protect against retinal capillary closure and had inconsistent effects on diabetes-related upregulation of ECM mRNAs. These results indicate that the AGE/ALE inhibitor PM protected against a range of pathological changes in the diabetic retina and may be useful for treating diabetic retinopathy.

Dietary antioxidant and mineral intake in humans is associated with reduced risk of esophageal adenocarcinoma but not reflux esophagitis or Barrett's esophagus

The role of antioxidants in the pathogenesis of reflux esophagitis (RE), Barrett's esophagus (BE), and esophageal adenocarcinoma (EAC) remains unknown. We evaluated the associations among dietary antioxidant intake and these diseases. We performed an assessment of dietary antioxidant intake in a case control study of RE (n = 219), BE (n = 220), EAC (n = 224), and matched population controls (n = 256) (the Factors Influencing the Barrett's Adenocarcinoma Relationship study) using a modification of a validated FFQ. We found that overall antioxidant index, a measure of the combined intake of vitamin C, vitamin E, total carotenoids, and selenium, was associated with a reduced risk of EAC [odds ratio (OR) = 0.57; 95% CI = 0.33-0.98], but not BE (OR = 0.95; 95% CI = 0.53-1.71) or RE (OR = 1.60; 95% CI = 0.86-2.98), for those in the highest compared with lowest category of intake. Those in the highest category of vitamin C intake had a lower risk of EAC (OR = 0.37; 95% CI = 0.21-0.66; P-trend = 0.001) and RE (OR = 0.46; 95% CI = 0.24-0.90; P-trend = 0.03) compared with those in the lowest category. Vitamin C intake was not associated with BE, and intake of vitamin E, total carotenoids, zinc, copper, or selenium was not associated with EAC, BE, or RE. In conclusion, the overall antioxidant index was associated with a reduced risk of EAC. Higher dietary intake of vitamin C was associated with a reduced risk of EAC and RE. These results suggest that antioxidants may play a role in the pathogenesis of RE and EAC and may be more important in terms of progression rather than initiation of the disease process.

Effect of phytoestrogen and antioxidant supplementation on oxidative DNA damage assessed using the comet assay

Antioxidant species may act in vivo to decrease oxidative damage to DNA, protein and lipids thus reducing the risk of coronary heart disease and cancer. Phytoestrogens are plant compounds which are a major component of traditional Asian diets and which may be protective against certain hormone-dependent cancers (breast and prostate) and against coronary heart disease. They may also be able to function as antioxidants, scavenging potentially harmful free radicals. In this study, the effects of the isoflavonoids (a class of phytoestrogen) genistein and equol on hydrogen peroxide-mediated DNA damage in human lymphocytes were determined using alkaline single-cell gel electrophoresis (the comet assay). Treatment with hydrogen peroxide significantly increased the levels of DNA strand breaks. Pre-treatment of the cells with both genistein and equol offered protection against this damage at concentrations within the physiological range. This protection was greater than that offered by addition of the known antioxidant vitamins ascorbic acid and alpha -tocopherol, or the compounds 17 beta -oestradiol and Tamoxifen which have similar structures to isoflavonoids and are known to have weak antioxidant properties. These findings are consistent with the hypothesis that phytoestrogens can, under certain conditions, function as antioxidants and protect against oxidatively-induced DNA damage. (C) 2001 Elsevier Science B.V. All rights reserved.

Endothelial Nox4 NADPH oxidase enhances vasodilatation and reduces blood pressure in vivo.

OBJECTIVE: Increased reactive oxygen species (ROS) production is involved in the pathophysiology of endothelial dysfunction. NADPH oxidase-4 (Nox4) is a ROS-generating enzyme expressed in the endothelium, levels of which increase in pathological settings. Recent studies indicate that it generates predominantly hydrogen peroxide (H(2)O(2)), but its role in vivo remains unclear. METHODS AND RESULTS: We generated transgenic mice with endothelium-targeted Nox4 overexpression (Tg) to study the in vivo role of Nox4. Tg demonstrated significantly greater acetylcholine- or histamine-induced vasodilatation than wild-type littermates. This resulted from increased H(2)O(2) production and H(2)O(2)-induced hyperpolarization but not altered nitric oxide bioactivity. Tg had lower systemic blood pressure than wild-type littermates, which was normalized by antioxidants. CONCLUSION: Endothelial Nox4 exerts potentially beneficial effects on vasodilator function and blood pressure that are attributable to H(2)O(2) production. These effects contrast markedly with those reported for Nox1 and Nox2, which involve superoxide-mediated inactivation of nitric oxide. Our results suggest that therapeutic strategies to modulate ROS production in vascular disease may need to separately target individual Nox isoforms.

The effect of lutein- and zeaxanthin-rich foods v. supplements on macular pigment level and serological markers of endothelial activation inflammation and oxidation pilot studies in healthy volunteers:pilot studies in healthy volunteers

The aim of the present study was to compare the effect of lutein- and zeaxanthin-rich foods and supplements on macular pigment level (MPL) and serological markers of endothelial activation, inflammation and oxidation in healthy volunteers. We conducted two 8-week intervention studies. Study 1 (n 52) subjects were randomised to receive either carrot juice (a carotene-rich food) or spinach powder (a lutein- and zeaxanthin-rich food) for 8 weeks. Study 2 subjects (n 75) received supplements containing lutein and zeaxanthin, ß-carotene, or placebo for 8 weeks in a randomised, double-blind, placebo-controlled trial. MPL, serum concentrations of lipid-soluble antioxidants, inter-cellular adhesion molecule 1, vascular cell adhesion molecule 1, C-reactive protein and F2-isoprostane levels were assessed at baseline and post-intervention in both studies. In these intervention studies, no effects on MPL or markers of endothelial activation, inflammation or oxidation were observed. However, the change in serum lutein and zeaxanthin was associated or tended to be associated with the change in MPL in those receiving lutein- and zeaxanthin-rich foods (lutein r 0.40, P = 0.05; zeaxanthin r 0.30, P = 0.14) or the lutein and zeaxanthin supplement (lutein r 0.43, P = 0.03; zeaxanthin r 0.22, P = 0.28). In both studies, the change in MPL was associated with baseline MPL (food study r - 0.54, P <0.001; supplement study r - 0.40, P <0.001). We conclude that this 8-week supplementation with lutein and zeaxanthin, whether as foods or as supplements, had no significant effect on MPL or serological markers of endothelial activation, inflammation and oxidation in healthy volunteers, but may improve MPL in the highest serum responders and in those with initially low MPL.

Lycopene intervention reduces inflammation and improves HDL functionality in moderately overweight middle-aged individuals

The management of overweight subjects by interventions aimed at reducing inflammation is highly desirable. To date, observational studies have identified a link between increased dietary antioxidant intake and reduced cardiovascular morbidity. However, direct trial evidence regarding the ability of antioxidants to influence inflammation is lacking. Therefore, this study examined lycopene's ability to lower systemic and high-density lipoprotein (HDL)-associated inflammation in moderately overweight middle-aged subjects. Serum was collected before and after a 12-week intervention from 54 moderately overweight, middle-aged individuals. Subjects were randomised to one of three groups: control diet (

Mitochondrial J haplogroup is associated with lower blood pressure and anti-oxidant status: findings in octo/nonagenarians from the BELFAST Study

Mitochondria produce cellular energy but also free-radicals, which damage cells despite an array of endogenous anti-oxidants. In Northern Europe, the mitochondrial haplogroup J has been related to longevity in nonagenarians and centenarians but also with age-related disease. Hypertension is an important contributor to atherosclerotic-related diseases and its pathogenesis is associated with increased oxidative stress. In this study, we questioned whether J haplogroup octo/nonagenarians from the Belfast Elderly Longitudinal Free-living Elderly STudy (BELFAST) study showed evidence of protective blood pressure or anti-oxidant profile which might explain their longevity advantage. Briefly, in a cross-sectional study, community-living, mentally alert (Folstein >25/30), octo/nonagenarian subjects, recruited for good health, were enlisted and consented as part of the BELFAST study, for blood pressure, anthropometric measurements and blood sampling. DNA typing for mitochondrial haplotypes was carried out with measurements for enzymatic and non-enzymatic antioxidants. J haplogroup carriers showed lower systolic blood pressure and glutathione peroxidase activity (Gpx) with higher folate measurements. There was no change in urate, bilirubin, albumin or nutrition-related antioxidants-selenium or vitamins A, C and a and ß carotene. BELFAST study mtDNA J haplogroup octo/nonagenarians showed lower blood pressure and reduced glutathione peroxidase activity and higher folate, but no change for other antioxidants. These findings are of interest in view of mtDNA J haplogroup's association with increased age in some previous studies.