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Chronic kidney disease patient's progression to end-stage renal disease as well as their high mortality are linked to cardiovascular disease. However, the high incidence rate of cardiovascular morbidity and mortality in these patients is not fully accounted for by traditional cardiovascular risk factors such as diabetes, hypertension and obesity. Renal disease and CVD are associated with endothelial dysfunction, inflammation and oxidative stress and in this review we will examine what is known regarding their similar roles in both CVD and chronic kidney disease, specifically focusing on the interconnections between oxidative stress, inflammation and endothelial dysfunction. These interconnections are best visualized as a vicious circle wherein these entities coexist and communicate with each other, thereby exacerbating the processes underpinning these different entities with the end result of the high morbidity and mortality that characterize CKD patients. By exploring this vicious circle i.e. the mode and extent of the interrelationships as well as some of the underlying mechanisms involved, this review aims at outlining our current understanding as well as highlighting future avenues for research and potential targets for therapeutic intervention., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Fabry disease is characterized by deficient expression/activity of α-GalA with consequent lysosomal accumulation in various organs of its substrate Gb3. Despite enzyme replacement therapy, Fabry disease progresses with serious myocardial, cerebral and renal manifestations. Gb3 accumulation may induce oxidative stress (OxSt), production of inflammatory cytokines and reduction of nitric oxide, which may impact on Fabry disease's clinical manifestations., Methods: OxSt status was characterized in 10 patients compared with 10 healthy subjects via protein expression of p22phox, subunit of NADH/NADPH oxidase, (Western blot), Heme oxygenase (HO)-1 levels (ELISA), antioxidant/anti-inflammatory, lipid peroxidation as malondialdehyde (MDA) production (colorimetric assay), phosphorylation state of Extracellular Signal Regulated Kinase (ERK)1/2 and Myosin Phosphatase Target Protein (MYPT)-1 (Western blot), marker of Rho kinase activation, both involved in OxSt signaling. Cardiac left ventricular (LV) mass was also evaluated (M-mode echocardiography)., Results: LV mass was higher in Fabry's males (123.72±2.03SEM g/m2) and females (132.09±6.72g/m2). p22phox expression was also higher in patients (1.04±0.09 d.u. vs 0.54±0.05 d.u. p<0.01) as well as MDA levels (54.51±3.97 vs 30.05±7.11 nmol/mL p = 0.01) while HO-1 was reduced (8.84±0.79 vs 14.03±1.23 ng/mL, p<0.02). MYPT-1's phosphorylation was increased in patients (0.52±0.11 d.u. vs 0.03±0.08 d.u., p<0.01) while phosphorylation of ERK1/2 was reduced (0.91±0.08 d.u. vs 1.53±0.17 d.u., p = 0.004)., Conclusions: This study documents OxSt activation and the altered reaction to it in Fabry patients. Cardiac remodeling, Rho kinase signaling activation and reduction of protective HO-1 might suggest that, in addition to enzyme replacement therapy, OxSt inhibition by either pharmacological or nutritional measures, is likely to prove useful for the prevention/treatment of Fabry patients' cardiovascular-renal remodeling., Competing Interests: This study was supported by an unrestricted grant from Sanofi- Genzyme. Linda Landini is employed by Sanofi-Genzyme. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Background & Aims: Cardiovascular disease, the most common cause for morbidity and mortality in end-stage renal disease (ESRD), has prompted the exploration of multiple approaches to improve outcomes. Cardiovascular risk factors such as oxidative stress (OxSt) and cardiac remodelling are common in ESRD and dialysis patients. Green tea (GT) is well recognized as reducing OxSt. This 6 months study evaluated in 20 ESRD patients under chronic dialysis, the effect of GT treatment (1 g/day as commercially available capsule) on cellular and plasma OxSt and proliferation related markers using a molecular biology approach., Methods: Mononuclear cell p22(phox), Haeme Oxygenase (HO)-1 protein expression, and phosphorylated ERK1/2 status were evaluated in dialysis patients at baseline, after 3 and 6 months of GT treatment by Western blot analysis and plasma oxLDL by ELISA. Cardiac remodelling was assessed by echocardiographic left ventricular (LV) mass determination at baseline and at the end of the study., Results: GT treatment reduced p22(phox) and pERK1/2 from baseline while HO-1 increased. At baseline, LV mass correlated with both p22(phox) and oxLDL. GT treatment decreased LV mass from baseline, which correlated with oxLDL. 9 patients had LV hypertrophy at baseline, which, at 6 months, was normalized in 5 and reduced in 3, showing a parallel decrease of p22(phox), pERK1/2, oxLDL and increase of HO-1., Conclusions: Treatment with GT decreased the expression of OxSt-related proteins tightly associated with cardiovascular disease and decreased LV mass. It appears highly likely that the addition of GT can provide a benefit in terms of cardiovascular protection in dialysis patients., (Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

The role of oxidative stress in the pathophysiology of hypertension and target organ damage is widely recognized. Using a molecular biology approach, we report, in essential hypertensive patients, the effect of the angiotensin II type 1 receptor blocker olmesartan on the mononuclear cell (PBMC) protein expression of major elements in the oxidative stress and vascular remodeling-related pathways, p22(phox) and HO-1, along with the phosphorylation state of ERK1/2 and plasma oxidized low-density lipoproteins (oxLDL). Twenty untreated essential hypertensive patients (range blood pressure: 142?156/94?98 mmHg) were treated with olmesartan medoxomil (20 mg/day for 6 months) and blood samples collected at baseline, 3 and 6 months for PBMC p22(phox) and HO-1 protein expression, phosphorylation state of ERK1/2 (western blot) and oxLDL level (ELISA) evaluations. Olmesartan normalized blood pressure since the third month (149 ? 4.7/94.88 ? 1.9 mmHg vs 137.89 ? 2.08/88.44 ? 2.0 at 3 months and vs 135.44 ? 2.18/85.78 ? 1.2 at 6 months, analysis of variance: p < 0.001). p22(phox) protein level declined at 3 months (7.10 ? 2.61 vs 9.32 ? 2.43 densitometric units (d.u.; p < 0.001), further declining at 6 months (4.55 ? 1.26 d.u., p < 0.001). HO-1 levels increased at 3 months (10.87 ? 1.92 vs 7.70 ? 0.71 d.u., p = 0.001) and remained elevated (11.11 ? 1.89 d.u., p = 0.001), without further increase at 6 months. Phosphorylated ERK1/2 declined at 3 months (3.94 ? 1.44 vs 5.62 ? 1.11 d.u., p = 0.001), further declining at 6 months (1.94 ? 0.87, p < 0.001). oxLDL significantly declined at 3 and 6 months. These results demonstrate that olmesartan inhibits oxidative stress. Given the involvement of oxidative stress and its signaling in atherogenesis, and the available evidence of olmesartan's vasoprotective, anti-inflammatory and antiatherosclerotic effects derived from clinical trials in humans, the results of our study provide a mechanistic rationale for the omelsartan's antioxidant and anti-inflammatory potential translation, in the long term, toward the antiatherosclerotic and antiremodeling effects reported on the clinical ground.

Hemodiafiltration with regeneration of ultrafiltrate (HFR) has a positive impact on inflammation and oxidative stress (OxSt), risk factors for cardiovascular disease (CVD), the most common cause of excess morbidity and mortality for end-stage renal disease (ESRD) patients. However, studies have been of limited duration. This study extends our previous study of HFR effects by evaluating the effect on mononuclear cell protein expression of heme-oxygenase-1 (HO-1), induced by OxSt, and inducible subunit of nitric oxide synthase (iNOS), and plasma level of interleukin-1β (Il-1β) and oxidized low-density lipoproteins (OxLDL), marker of OxSt, for a 12-month period. Fourteen ESRD patients stable on hemodialysis over a period of at least 2 years and on conventional bicarbonate dialysis were switched to be treated with HFR. Blood samples were collected at baseline, after 3, 6, 9 and 12 months. HO-1 and iNOS protein expression were evaluated by Western blot, OxLDL by enzyme-linked immunosorbent assay (ELISA), and Il-1β by enzyme amplified sensitivity immumoassay assay. HFR significantly increased HO-1 at the 9 and 12 months (ANOVA = P < 0.00001): 0.17 ± 0.11 (baseline) versus 0.48 ± 0.20, P < 0.043 and 0.59 ± 0.32, P < 0.004, respectively. Il-1β declined (ANOVA = P < 0.0001) since the 3 months from 169.92 ± 92.39 pg/mL (baseline) to 39.03 ± 10.01 (12 months), P < 0.0001. HFR also reduced plasma OxLDL: 475.4 ± 110.8 ng/mL (baseline) versus 393.1 ± 101.9 ng/mL (12 months), P < 0.04. iNOS showed no changes upon HFR treatment. These results together with our previous results indicate that HFR improves OxSt and inflammation. Given the strong relationships between OxSt and inflammation with CVD, their reduction might provide a beneficial impact by reducing the risk of atherosclerotic CVD in dialysis patients., (© 2010, Copyright the Authors. Artificial Organs © 2010, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Background and Aim: Angiotensin II (Ang II) induces oxidative stress (OxSt), which is essential for cardiovascular remodeling. Aldosterone also induces fibrosis and remodeling through direct effect on non-classical mineralocorticoid (MR) target tissues. However, studies on the role of aldosterone on OxSt and related factors in humans are lacking., Materials and Methods: We assessed gene and protein expression of p22phox (RT-PCR and Western blot), NAD(P)H oxidase subunit essential for superoxide production and gene expression of transforming growth fator (TGF) beta, plasminogen activator inhibitor (PAI)-1, and heme oxygenase (HO)-1, effectors of OxSt (RT-PCR), in a Conn's adenoma, removed from a patient with primary hyperaldosteronism. Ang II type 1 (AT1R) and MR receptors expression were also evaluated (RT-PCR). The normal adrenal tissue adjacent to the adenoma was used as control., Results: p22phox gene and protein expression were higher (31% and 53%, respectively) in the adrenal adenoma. TGFbeta, PAI-1, and HO-1 gene expression were also higher (25%, 129%, and 25%, respectively) in the adrenal adenoma while AT1R gene expression was similar (8%). The expression of MR in the adenoma was documented., Conclusions: This report demonstrates in a human model that the increased aldosterone production has effects on enzyme systems related to OxSt, enhancing the systemic fibrogenic effects of aldosterone excess through TGFbeta and PAI-1 expression which was previously demonstrated only indirectly in vitro and in animal models. The presence of MR expression in the adenoma may link the hormone with the adenoma growth. Therefore, the results of this study derived from a single case might represent an important working hypothesis for further research in a larger number of cases to clarify the role of aldosterone overproduction on OxSt and its clinical relevance.

Objective: Hyperglycemia is the main determinant of long-term diabetic complications, mainly through induction of oxidative stress. NAD(P)H oxidase is a major source of glucose-induced oxidative stress. In this study, we tested the hypothesis that rosiglitazone (RSG) is able to quench oxidative stress initiated by high glucose through prevention of NAD(P)H oxidase activation., Methods and Results: Intracellular ROS were measured using the fluoroprobe TEMPO-9-AC in HUVECs exposed to control (5 mmol/L) and moderately high (10 mmol/L) glucose concentrations. NAD(P)H oxidase and AMPK activities were determined by Western blot. We found that 10 mmol/L glucose increased significantly ROS production in comparison with 5 mmol/L glucose, and that this effect was completely abolished by RSG. Interestingly, inhibition of AMPK, but not PPARgamma, prevented this effect of RSG. AMPK phosphorylation by RSG was necessary for its ability to hamper NAD(P)H oxidase activation, which was indispensable for glucose-induced oxidative stress. Downstream of AMPK activation, RSG exerts antioxidative effects by inhibiting PKC., Conclusions: This study demonstrates that RSG activates AMPK which, in turn, prevents hyperactivity of NAD(P)H oxidase induced by high glucose, possibly through PKC inhibition. Therefore, RSG protects endothelial cells against glucose-induced oxidative stress with an AMPK-dependent and a PPARgamma-independent mechanism.

Background: Increased oxidative stress (OxSt) as well as inflammation are risk factors for cardiovascular events and determinant of cardiovascular disease which remains the most common cause of excess morbidity and mortality for end-stage renal disease ESRD patients. Haemodiafiltration with on-line regeneration of ultrafiltrate (HFR) has been shown to have a positive impact on markers of inflammation while its effect on OxSt is not known., Methods: This study evaluates in haemodialysis patients the effect of HFR on the plasma level of oxidized LDL (OxLDL), a marker of OxSt, and mononuclear cell gene and protein expression of OxSt-related proteins such as p22phox (subunit of NAD(P)H oxidase), PAI-1 (induced by OxSt and atherothrombogenetic) and haeme-oxygenase-1 (HO-1) (induced by OxSt). Fourteen patients were randomized into two groups in a crossover design, treated for 6 month periods with HFR (SG8 Plus-Bellco, Mirandola, Italy) or low-flux bicarbonate dialysis (HD) using a polysulphone dialyser 1.8 m2. Blood samples were collected at the beginning of the study, after 6 months (crossover) and after 12 months., Results: ANOVA analysis of the data performed to rule out any crossover effect in either sequence was not significant and thus data from both sequences were combined and then analysed further statistically. HFR reduced mRNA production and protein expression of p22phox and PAI-1 compared with HD (-9+/-5 vs 2+/-6 Delta%, P<0.0001 and -15+/-20 vs 3+/-17 Delta%, P<0.05 for p22phox; -19+/-6 vs -5+/-5 Delta%, P<0.0001 and -24+/-12 vs 9+/-15 Delta%, P<0.0001 for PAI-1). HO-1 was unchanged (-12+/-8 vs -10+/-8 Delta% and -21+/-12 vs -14+/-8 Delta%) while plasma OxLDL was reduced (-14+/-19 vs 1+/-14 Delta%, P<0.01)., Conclusions: The results of our study indicate that HFR treatment, compared with standard dialysis, has a lower impact on OxSt. Given, the strong relationship between OxSt and inflammation and their impact on the long-term cardiovascular complications in end-stage renal disease patients, HFR might have a more beneficial impact in reducing the risk of atherosclerotic cardiovascular disease in dialysis patients.

Objective: To test the hypothesis that oxidative stress signaling contributes to post-transplant endothelial dysfunction and hypertension in pediatric post-transplant hypertension., Study Design: This study evaluated in 16 pediatric renal transplant patients, divided in two groups based on the presence of post-transplant hypertension, the oxidative stress status measuring the gene expression (reverse transcription-polymerase chain reaction) of two major oxidative stress-related proteins, p22(phox) and heme oxygenase-1 (HO-1). Total plasma antioxidant power (ELISA) was also evaluated., Results: Mononuclear cell p22(phox) gene expression was higher in hypertensive patients compared with the normotensive group (0.91 +/- 0.06 vs 0.79 +/- 0.08 densitometric units, P < .02), whereas HO-1 RNA production and total plasma antioxidant power were higher in the normotensive group (0.38 +/- 0.04 vs 0.20 +/- 0.11 d.u., P < .006, and 1189.35 +/- 145.75 vs 772.71 +/- 196.03 micromol/L, P < .01, respectively)., Conclusions: Oxidative stress is associated with post-transplant hypertension in hypertensive pediatric kidney-transplant patients, who therefore are at risk of oxidative stress-induced organ damage. They might benefit from treatments addressing not only hypertension but also oxidant-related complications.

Background: Increased oxidative stress is associated with all cardiovascular risk factors and reactive oxygen species appear to be the principal mediators of cardiomyocite dysfunction in various cardiovascular diseases. Carnitine has been shown to be effective in pathologic conditions characterized by increased oxidative stress and an antioxidant effect of L-carnitine and its derivatives has been described but the specific mechanism is unclear., Methods: We evaluated in human endothelial cells in culture the effect of L-carnitine (C), acetyl-L-carnitine (AC) and propionyl-L-carnitine (PC) on gene and protein expression (RT-PCR and Western blot) of oxidative stress related proteins heme oxygenase-1 (HO-1) and of endothelial NO synthase (ecNOS) in absence and presence of oxidative stress induced by H2O2., Results: HO-1 as well as ecNOS gene and protein expression significantly increased upon Carnitines incubation. Induction of oxidative stress increased HO-1 gene expression compared to basal condition (0.62+/-0.02 densitometric units vs. 0.48+/-0.05, p<0.01) while decreased ecNOS gene expression (0.75+/-0.04 vs. 0.40+/-0.08, p<0.001). These results were paralleled by similar results at protein level. Coincubation of C (0.5-1.0-2.0 mM), AC (0.1-0.2-0.4 mM) and PC (0.05-0.1-0.2 mM) with H2O2 further increased HO-1 gene expression and not only normalized vs. H2O2 but even increased vs. basal ecNOS mRNA. HO-1 and ecNOS gene expression was also paralleled at protein level by coincubation with C, AC and PC of cells exposed to oxidative stress., Conclusion: This is the first report that has utilized a molecular biological approach to demonstrate a direct stimulatory effect of Carnitines on gene and protein expression of the oxidative stress related markers HO-1 and ecNOS. As HO-1 and NO are known as antioxidant, antiproliferative and anti-inflammatory, their increased expression would be expected to protect from oxidative stress related cardiovascular risk factors and myocardial damage, therefore adding this effect to the multiple pathways involved in the effects of carnitines.

The role of oxidative stress in the pathophysiology of hypertension has stimulated the investigation of strategies to reduce oxidative stress via antioxidant defenses. Using a molecular biology approach, we report, in essential hypertensive patients, the effect of doxazosin treatment on the mononuclear cell gene and protein expression of two major elements in the oxidative stress and vascular remodeling-related pathways: p22(phox) and PAI-1. Ten essential hypertensive patients were treated with Doxazosin (4 mg/day) for two weeks (EH + D) and compared with ten untreated hypertensive patients (EH) and ten normotensive subjects (C). In EH p22(phox) and PAI-1 mRNA and protein level was increased compared with C. In EH + D, doxazosin reduced p22(phox) and PAI-1 gene and protein expression, which was similar to that of C. These results demonstrate for doxazosin an inhibitory effect on oxidative stress related proteins at gene and protein level, which confirms at molecular level a powerful antioxidant potential for this agent that could translate, in the long term, into a powerful antiatherosclerotic effect.

Background and Objectives: Oxidative stress can induce cell mutations or proliferation which then can progress to carcinogenesis or remodeling. The same oxidative stress-mediated mechanism could participate in prostate cell proliferation and remodeling present in benign prostatic hyperplasia (BPH). Doxazosin induces prostate epithelial and stromal cell apoptosis through production of transforming growth factor-beta (TGF-beta), but cellular mechanisms are not completely clarified. In 10 prostate samples from BPH untreated patients who underwent TUR, we have assessed the gene and protein expression of: p22(phox) (subunit of NAD(P)H oxidase essential for O(2)(-) production); heme oxygenase-1 (HO-1) (induced by oxidative stress and antiapoptotic); TGF-beta (inhibitor of prostatic epithelial and stromal cell growth); the in vitro effect of doxazosin on expression of these markers., Methods: RT-PCR and Western blot with specific primers and antibodies. p22(phox), HO-1 and TGF-beta were quantified by the ratio between their PCR and Western blot products and GAPDH., Results: Doxazosin significantly reduced p22(phox) gene and protein expression (0.61 +/- 0.04 vs. 0.36 +/- 0.04 d.u., p < 0.0002; 0.85 +/- 0.03 vs. 0.47 +/- 0.03, p < 0.0001, respectively). Doxazosin concentration dependently reduced HO-1 gene and protein expression (0.57 +/- 0.07 vs. 0.49 +/- 0.06 d.u. (1 microM) p < 0.04, vs. 0.22 +/- 0.08 (10 microM) p < 0.0001; 0.78 +/- 0.04 vs. 0.44 +/- 0.1 (10 microM) p < 0.003 respectively) and increased TGF-beta protein expression (0.58 +/- 0.05 vs. 0.74 +/- 0.16 (1 microM) n.s. vs. 0.81 +/- 0.07 (10 microM) p < 0.01)., Conclusions: Induction of oxidative stress-related proteins seems to be involved in the prostate cell proliferation and remodeling present in BPH. Doxazosin may reduce oxidative stress through reduction of p22(phox). Surprisingly, HO-1, which is induced and protected by oxidative stress, is also reduced by doxazosin. HO-1 is a potent antiapoptotic factor and downregulator of TGF-beta. From the results of this preliminary study it could be proposed that the proapoptotic effect of doxazosin could be mediated, at least in part, through the contemporary inhibition of HO-1., (Copyright (c) 2006 S. Karger AG, Basel.)

Oxidative stress plays a role in cardiovascular dysfunction. This is of interest in diabetes, a clinical condition characterized by oxidative stress and increased prevalence of cardiovascular disease. The role of p66(shc) in oxidative stress-related response has been demonstrated by resistance to and reduction of oxidative stress and prolonged lifespan in p66(shc-/-) mice. In this study we assess p66(shc) gene expression in peripheral blood mononuclear cells (PBM) from type 2 diabetic patients and healthy subjects. The p66(shc) mRNA level was assessed using RT-PCR with two sets of primers mapping for different p66(shc) regions. p66(shc) is expressed in both monocytes and lymphocytes. The level of p66(shc) mRNA was significantly higher in type 2 diabetic patients compared with controls (0.38 +/- 0.07 densitometric units vs. 0.13 +/- 0.08; P < 0.0001). In addition, total plasma 8-isoprostane levels, a marker of oxidative stress, were higher in type 2 diabetics (0.72 +/- 0.04 ng/ml) than in normal subjects (0.43 +/- 0.04, P < 0.001) and were significantly correlated to the p66(shc) mRNA level in PBM from type 2 diabetics (r(2) = 0.47; P = 0.0284). In conclusion, diabetes induces p66(shc) gene expression in circulating PBM; this up-regulation in expression is significantly associated with markers of oxidative stress. p66(shc) gene expression in PBM may represent a useful tool to investigate the oxidative stress involved in the pathogenesis of long-term diabetic complications.

Background: Hemodialysis patients (HD) are exposed to oxidative stress which contributes to cardiovascular disease and accelerated atherosclerosis, major causes of mortality in these patients. A new dialysis membrane coated with vitamin E has been proposed against oxidative stress and atherosclerosis due to their ability to inhibit lipid peroxidation by interacting with scavengers. The mechanisms however are not completely clarified. This study evaluated, using a molecular biology approach, the effect of 6 months treatment with vitamin E-modified dialyzers, CL-E, on the gene expression of oxidative stress related proteins and markers., Patients and Methods: To this end, the gene expression of p22phox, a NAD(P)H oxidase subunit closely linked with the generation of superoxide anions and of Heme oxygenase-1 (HO-1), induced by and protective from oxidative stress, were evaluated by RT-PCR in mononuclear cells from 5 patients under 3 times a week chronic bicarbonate dialysis. Hydroperoxide (HPO) and total antioxidant power (AOP) plasma levels were evaluated at 3 and 6 months of treatment. HPO was also evaluated in 8 patients under CL-E treatment for 1 year and compared with 8 patients treated with cuprammonium-ryon filter (TAF)., Results: p22phox mRNA decreased from 0.61 +/- 0.05 d.u. to 0.48 +/- 0.03, p < 0.01 while HO-1 increased from 0.55 +/- 0.04 d.u. to 0.62 +/- 0.03, p < 0.01. HPO decreased in CL-E treated patients: from 2.72 +/- 0.26 microM to 1.45 +/- 0.27 at 3 months (p < 0.001) to 0.87 +/- 0.11, p < 0.001 at 6 months, while AOP increased: from 752 +/- 90 mmol/L to 1057 +/- 105, p < 0.001 at 6 months. HPO was also reduced in 1 year Excebrane CL-E treated patients compared with cuprammonium treated patients: 2.25 +/- 0.3 vs. 1.42 +/- 0.11 microM, p < 0.001., Conclusion: The reduced expression of oxidative stress related proteins and markers gives further support to the efficacy of the use of Vitamin E coated dialysers for the prevention or slowing progression of cardiovascular disease and atherosclerosis, major complications and causes of mortality in these patients in which oxidative stress plays a pivotal role.

Oxidative stress (OxSt) is a major damaging factor in arterial hypertension and its long-term complications. This is why considerable attention is paid to the possible effects of antihypertensive drugs on OxSt. Manidipine is a dihydropiridine calcium channel blocker with reported nephroprotective activities, but no information is available on its effect on OxSt and related mechanisms. This study assessed the effect of manidipine on normal subjects' monocyte gene and protein expression of OxSt-related proteins such as p22(phox), a NAD(P)H oxidase system subunit, critical in generating O2-, and heme oxygenase-1 (HO-1), induced by and protective from OxSt, and compared manidipine with the ACE inhibitor captopril and the calcium channel blocker nifedipine, in the presence and absence of sodium arsenite (NaAsO2) as an inducer of OxSt.Co-incubation of manidipine with NaAsO2 dose-dependently decreased p22(phox) mRNA production from basal: 0.87 +/- 0.1 d.u., 0.69 +/- 0.06 and 0.66 +/- 0.09 at 100, 300 and 500 nM respectively versus 0.99 +/- 0.2, P < 0.04, while HO-1 mRNA production was increased by the same concentrations of the drug: 0.87 +/- 0.1 d.u., 0.92 +/- 0.1, 0.98 +/- 0.1 respectively versus 0.63 +/- 0.07; P < 0.03. Monocyte p22(phox) mRNA production was reduced both by manidipine and captopril: 0.48 +/- 0.04 d.u. and 0.43 +/- 0.08, respectively versus 0.58 +/- 0.07, P < 0.006, while no changes were induced by nifedipine (0.61 +/- 0.07, P = ns). Manidipine increased monocyte HO-1 mRNA production (1.6 +/- 0.4 versus 1.2 +/- 0.4, P < 0.008), while nifedipine and captopril showed no effect (1.2 +/- 0.3 and 1.1 +/- 0.3, respectively). The effects of M on p22(phox) and HO-1 gene expression in the presence of OxSt were also paralleled by the same effects at protein level. In conclusion, manidipine decreases p22(phox) and increases HO-1 mRNA production and protein level. The manidipine-induced increase of HO-1 gene and protein expression seems to be a peculiar effect of this drug since it is not observed with captopril and nifedipine. This effect, together with the reduction of p22(phox) mRNA production, could play a role in its protective mechanism against OxSt.

Background: Bartter's and Gitelman's syndromes (BS/GS) have a blunted Gq protein-mediated cell signalling despite high circulating angiotensin II (Ang II) levels. This is associated with reduced Galphaq gene expression, intracellular inositol trisphosphate and Ca(++) release, PKC activity and cell reactivity. Ang II is a powerful stimulator of vascular oxidases but BS/GS patients show reduced total volatile LDL oxidation products and reduced LDL susceptibility to oxidation suggesting low level of oxidative stress. Therefore, we evaluated oxidative stress-related proteins in plasma and monocytes of patients with BS/GS, at baseline and after Ang II stimulation., Methods: In two BS and seven GS patients, biochemically and genetically characterized, and in 10 age- and sex-matched control subjects, we measured total plasma antioxidant power (AOP), plasma peroxynitrite level and gene expression of the NADH/NADPH oxidase subunit p22(phox), TGFbeta and haeme oxygenase-1 (HO-1) in circulating monocytes in basal condition and after stimulation with Ang II. Furthermore, we investigated the C(242)T polymorphism of p22(phox), whose topography in a potential haeme-binding site suggests a role in the regulation of oxidative stress., Results: AOP was higher in BS/GS patients than in controls (3.27 +/- 0.95 mmol/l vs 1.05 +/- 0.16, P = 0.002), together with higher plasma renin activity and aldosterone level (9.88 +/- 4.64 vs 0.95 +/- 0.08 nmol Ang I/h/ml, P < 0.0001; and 0.73 +/- 0.13 vs 0.18 +/- 0.01 nmol/l, P < 0.0001, respectively). The plasma peroxynitrite level was undetectable both in patients and controls. mRNA expression of p22(phox) and TGFbeta was reduced in BS/GS patients compared to controls [0.35 +/- 0.08 vs 0.53+/-0.05 densitometric units (d.u.), P = 0.005, and 0.82 +/- 0.07 vs 1.15 +/- 0.25 d.u., P = 0.006, respectively]. HO-1 mRNA was increased in BS/GS patients in comparison to controls (0.88 +/- 0.07 vs 0.78 +/- 0.11 d.u., P = 0.037). After acute Ang II exposure, p22(phox), TGFbeta and HO-1 gene expression significantly increased only in controls (from 0.59 +/- 0.12 to 0.96 +/- 0.11, P < 0.001, from 0.97 +/- 0.1 to 1.27 +/- 0.22, P < 0.008, and from 0.62 +/- 0.1 to 0.82 +/- 0.09, P < 0.001, respectively). Finally, C(242)T polymorphism of p22(phox) was undetectable., Conclusions: The intracellular responses to Ang II mediated by reactive oxygen species are reduced in BS/GS patients. This may contribute to their vascular hyporeactivity.

Ozone (O(3)) is among the most reactive environmental oxidant pollutants to which cutaneous tissues are exposed. O(3) exposure has been shown to induce antioxidant depletion as well as the oxidation of lipids and proteins within the outermost skin layer, the stratum corneum. However, relatively little is known regarding the potential effects of O(3) on the cellular constituents of the underlying skin epidermis and dermis. In the present study, hairless mice exposed for 6 h to 0.8 ppm O(3) showed increases in lipid peroxidation, as quantitated by increases in 4-hydroxynonenal-protein adducts. O(3) exposure caused an induction of the stress proteins HSP27 and heme oxygenase-1 (HO-1), starting at 6 h and increasing up to 18 h after O(3) exposure. This was accompanied by an increase in matrix metalloproteinase-9 (MMP-9) mRNA and activity levels, indicative of possible injurious-reparative processes. Collectively, our data demonstrate that skin exposure to O(3) not only affects antioxidant levels and oxidation markers in the outermost stratum corneum layer, but also induces cellular stress responses in the deeper cellular layers of the skin.

Oxidative stress is associated with diabetes mellitus: a role of vascular NADPH oxidase as a source of superoxide has been demonstrated. We determined whether in type 2 diabetes mononuclear cells, NADPH oxidase and the inducible hemeoxygenase (HO-1) gene expressions are activated. In monocytes from 25 outpatients with type 2 diabetes, p22(phox) gene expression was higher (0.71 +/- 0.09 p22(phox)/beta-actin gene expression ratio) than that observed in 19 controls (0.56 +/- 0.09, P < 0.001). Similarly, HO-1 gene expression was significantly higher in diabetic patients (0.77 +/- 0.12 HO-1/beta-actin gene expression ratio) than in controls (0.41 +/- 0.14, P < 0.001). The p22(phox) and HO-1 gene expressions were also determined during (plasma glucose 363 +/- 40 mg/dl) and after (125 +/- 11 mg/dl) metabolic decompensation in 10 type 2 diabetic patients. The correction of the metabolic milieu was associated with a 19% +/- 3% (P < 0.01) and 30% +/- 3% (P < 0.01) decrease in the p22(phox) and HO-1 gene expressions, respectively. In a multivariate analysis, age was independently associated to p22(phox) gene expression in circulating monocytes in type 2 diabetics [13% (adjusted R(2)), P < 0.05]. Decompensated type 2 diabetes is associated with increased p22(phox) and HO-1 gene expressions in circulating monocytes; the metabolic normalization reduces but does not normalize this activation. These findings suggest that these cells, which play a crucial role in the earliest events of atherosclerotic lesion, are subjected to an increased oxidative stress.

In patients with cyclosporine-induced hypertension, upregulation of the nitric oxide system and oxidative stress were shown, which could induce hypertension, remodeling, and chronic rejection by increasing nitric oxide catabolism. However, it is still debated whether cyclosporine and tacrolimus exert a different action. The aim of the current study was to compare the effects of cyclosporine and tacrolimus on markers of oxidative stress and endothelial dysfunction in kidney transplant patients with posttransplant hypertension. Monocyte p22, a NADH/NADPH system subunit, transforming growth factor-beta (TGF-beta), heme oxygenase-1 (HO-1), and endothelial NOS gene expression were measured in 16 patients. Angiotensin II is a potent stimulator of oxidative stress and angiotensin-converting enzyme inhibition may blunt this effect. Therefore, the same parameters were measured before and after 2 months of treatment with ramipril (5 mg/d). At baseline, in cyclosporine-and tacrolimus-treated patients, p22 and TGF-beta mRNA were similarly increased in comparison with normotensive healthy controls (0.90 +/- 0.05 d.u. and 0.83 +/- 0.05 in cyclosporine, 0.89 +/- 0.07 and 0.84 +/- 0.05 in tacrolimus; 0.53 +/- 0.07 and 0.75 +/- 0.03 in controls, respectively; p < 0.001). Endothelial NOS mRNA was increased in cyclosporine-and tacrolimus-treated patients in comparison with controls (0.92 +/- 0.09, 0.96 +/- 0.04, and 0.37 +/- 0.05 respectively; p < 0.001), whereas no difference was found between patients and controls in HO-1 mRNA. Ramipril reduced blood pressure (from 140 +/- 11/91 +/- 7 mm Hg to 129 +/- 6/85 +/- 5 mm Hg in cyclosporine and from 138 +/- 7/92 +/- 7 mm Hg to 127 +/- 10/82 +/- 6 mm Hg in tacrolimus group; p < 0.02 with no difference between groups). Ramipril also reduced p22 (to 0.83 +/- 0.05 in cyclosporine, p < 0.03 and to 0.81 +/- 0.08 in tacrolimus; p < 0.01) and TGF-beta mRNA (to 0.72 +/- 01 in cyclosporine, p < 0.02, and to 0.73 +/- 0.05 in tacrolimus; p < 0.01) with no difference between groups, but it did not change HO-1 and ecNOS mRNA. Cyclosporine and tacrolimus induce a comparable oxidative stress in kidney transplant patients with posttransplant hypertension. The association of ramipril normalizes blood pressure and reduces the oxidative stress induced by both drugs.

Cyclosporin is a powerful stimulator of oxidative stress signaling, leading to TGFbeta production, NO degradation, endothelial dysfunction, hypertension and post-transplant nephropathy. Carvedilol, alpha1-beta-blocker with strong antioxidant activity, may interfere with this chain of events. Therefore, we measured monocyte ecNOS, TGFbeta and heme oxygenase-1 (HO-1) mRNA level and plasma nitrite/nitrate, 3-nitrotyrosine, an estimate of peroxynitrite, and total plasma antioxidant power in kidney-transplanted patients with post-transplant hypertension, before and after treatment with carvedilol, 25 - 50 mg o.d. orally for 4 months (n = 15). The dihydropyridine calcium channel blocker nifedipine (n = 10) was used as comparator antihypertensive drug. Blood pressure fell to a similar extent with both drugs. Carvedilol increased plasma antioxidant power and HO-1 mRNA and reduced 3-nitrotyrosine and TGFbeta mRNA levels, while the same was not observed with nifedipine. Monocyte ec NOS mRNA levels and plasma nitrite/nitrate were higher in the patients than in a normotensive healthy control group and were unaffected by either treatment. In conclusion, carvedilol reduces the oxidative stress and corrects the altered cellular signaling mediated by oxidative stress in CsA-induced post-transplant hypertension. Therefore, it may prevent long-term complications, such as endothelial dysfunction, fibrogenesis and post-transplant nephropathy by decreasing NO degradation and production of TGFbeta, a key fibrogenic cytokine, and by activating HO-1 production.

Cyclosporin-induced hypertension and endothelial dysfunction have been attributed to the effects of cyclosporin on factors controlling vasomotor tone. Endothelial nitric oxide (NO) regulates basal vasodilation, and an NO-mediated protective mechanism from cyclosporin-induced vasoconstriction has been proposed. In transplanted patients with cyclosporin-induced hypertension, we have recently demonstrated upregulation of the NO system and superoxide and free radical overproduction, which, by increasing NO metabolism, could induce hypertension, vascular remodeling and chronic rejection. In the present work, we have evaluated endothelial constitutive NO synthase (ecNOS), transforming growth factor beta and heme oxygenase-1 (protective against oxidative stress), mRNA production and plasma NO metabolites, peroxynitrite and antioxidant power in 15 kidney transplanted patients before and after 4 months of treatment with carvedilol alpha 1-beta-blocker and potent antioxidant. Our aim was to study the efficacy of the reduction of oxidative stress on complications such as endothelial dysfunction and fibrogenesis. Monocyte ecNOS and plasma NO metabolites remained higher versus those of control subjects and were unchanged by carvedilol, while antioxidant power and heme oxygenase-1 mRNA production increased. Peroxynitrite, as well as transforming growth factor beta mRNA, were reduced by carvedilol. It also normalized blood pressure. In conclusion, carvedilol reduces oxidative stress and normalizes blood pressure; ecNOS remains upregulated while mRNA for transforming growth factor beta, a key fibrogenic cytokine, is reduced by carvedilol, which seems to preserve protective mechanisms such as NO and heme oxygenase-1 against long-term complications of oxidative stress, e.g., endothelial dysfunction, fibrogenesis and chronic rejection.

Background: CsA-induced endothelial dysfunction and CsA-induced hypertension have been attributed to CsA effects on the endothelial-derived factors controlling vasomotor tone, but the mechanisms responsible are unclear. Endothelial nitric oxide (NO) is known to maintain a state of basal vasodilation and recently a NO mediated counterregulatory mechanism protective from CsA-induced vasoconstriction has been suggested., Patients and Methods: Our study evaluates ecNOS gene status and NO metabolites in kidney transplanted patients under chronic CsA treatment with CsA-induced hypertension. Since CsA increases superoxide production, which metabolizes NO, plasma hydroperoxides and peroxynitrite were also evaluated as index of the presence of "oxidative stress"., Results: Quantification of monocyte ecNOS mRNA and NO metabolites plasma level from patients and control subjects (C) demonstrated NO system up regulation in patients notwithstanding hypertension. The mean ecNOS to beta-actin ratio was 2.00 +/- 0.87 vs 0.29 +/- 0.08 in C, p < 0.04. NO metabolite plasma level was 30.03 +/- 9.62 mM vs 9.37 +/- 3.86, p < 0.001. Hydroperoxides were also increased in patients: 3.6 +/- 1.6 i.a.u. vs 1.4 +/- 0.8, p < 0.007 (from cholesterol esters) and 10.8 +/- 6.6 vs 1.5 +/- 0.9, p < 0.008 (from triglycerides) as well as peroxynitrite plasma level: 0.36+/- 0.14 mM/L vs undetectable in C., Conclusions: This study confirms a NO system up-regulation in transplanted patients. However, the counterregolatory system to CsA-induced vasoconstriction, could be cancelled by CsA induced superoxide and free radicals production which, increasing NO metabolism could contribute to CsA induced vasoconstriction and hypertension.