Oscar Beloqui

Superoxide Dismutase in Patients With Chronic Hepatitis C Virus Infection

It has been reported that hepatitis C virus (HCV) may cause oxidative stress in infected cells. Patients with chronic hepatitis C exhibit an increased production of tumor necrosis factor-alpha (TNF alpha), a cytokine that can produce oxidative stress by stimulating the generation of reactive oxygen species (ROS). Cell defense against ROS includes overexpression of Mn-superoxide dismutase (SOD), an inducible mitochondrial enzyme. To investigate cell defense against oxidative stress in HCV infection, we analyzed Mn-SOD mRNA in liver and in peripheral blood mononuclear cells (PBMC) from patients with chronic hepatitis C. Mn-SOD expression in PBMC was significantly increased in patients with HCV infection. Patients with sustained virological and biochemical response after therapy showed significantly lower Mn-SOD than patients with positive viremia. By contrast, Mn-SOD expression was not enhanced in the liver of patients with chronic hepatitis C. The values of Mn-SOD mRNA did not correlate with TNF alpha mRNA expression, viral load, or liver disease activity. Our results indicate that in HCV infection an induction of Mn-SOD was present in PBMC but absent in the liver, suggesting that this organ could be less protected against oxidative damage. Oxidative stress could participate in the pathogenesis of HCV infection.

Association of increased phagocytic NADPH oxidase-dependent superoxide production with diminished nitric oxide generation in essential hypertension

Objective Oxidative stress has been implicated in the pathogenesis of hypertension and its complications through alterations in nitric oxide (NO) metabolism. This study was designed to investigate whether a relationship exists between phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent superoxide anion (•O2−) production and NO generation in patients with essential hypertension. Methods Superoxide production was assayed by chemiluminescence under baseline and stimulated conditions on mononuclear cells obtained from hypertensives (n = 51) and normotensives (n = 43). NO production was evaluated by determining serum NO metabolites, nitrate plus nitrite (NOx). Results Although there were no differences in baseline •O2− production between normotensives and hypertensives, the •O2− production in phorbol myristate acetate (PMA)-stimulated mononuclear cells was increased (P < 0.05) in hypertensives compared with normotensives. The PMA-induced •O2− production was completely abolished by apocynin, a specific inhibitor of NADPH oxidase. Moreover, stimulation of •O2− production by angiotensin II and endothelin-1 was higher (P < 0.05) in cells from hypertensives than in cells from normotensives. In addition, diminished (P < 0.001) serum NOx was detected in hypertensives compared with normotensives. Interestingly, an inverse correlation (r = 0.493, P < 0.01) was found between •O2− production and NOx in hypertensives. Conclusions Generation of •O2− mainly dependent on NADPH oxidase is abnormally enhanced in stimulated mononuclear cells from hypertensives. It is suggested that this alteration could be involved in the diminished NO production observed in these patients.

Preliminary characterisation of the promoter of the human p22phox gene: identification of a new polymorphism associated with hypertension

The p22phox subunit is an essential protein in the activation of NAD(P)H oxidase. Here we report the preliminary characterisation of the human p22phox gene promoter. The p22phox promoter contains TATA and CCAC boxes and Sp1, γ‐interferon and nuclear factor κB sites. We screened for mutations in the p22phox promoter and identified a new polymorphism, localised at position −930 from the ATG codon, which was associated with hypertension. Mutagenesis experiments showed that the G allele had higher promoter activity than the A allele. These results suggest that the −930A/G polymorphism in the p22phox promoter may be a novel genetic marker associated with hypertension.

Functional Effect of the p22phox −930A/G Polymorphism on p22phox Expression and NADPH Oxidase Activity in Hypertension

Oxidative stress induced by superoxide is implicated in hypertension. NADPH oxidase is the main source of superoxide in phagocytic and vascular cells, and the p22phox subunit is involved in NADPH oxidase activation. Recently we reported an association of −930A/G polymorphism in the human p22phox gene promoter with hypertension. This study was designed to investigate the functional role of this polymorphism in hypertension. We thus investigated the relationships between the −930A/G polymorphism and p22phox expression and NADPH oxidase–mediated superoxide production in phagocytic cells from 70 patients with essential hypertension and 70 normotensive controls. Genotyping of the polymorphism was performed by restriction fragment length polymorphism. NADPH oxidase activity was determined by chemiluminescence assays, and p22phox mRNA and protein expression was measured by Northern and Western blotting, respectively. Compared with hypertensive subjects with the AA/AG genotype, hypertensive subjects with the GG genotype exhibited increased (P <0.05) phagocytic p22phox mRNA (1.26±0.06 arbitrary unit [AU] versus 0.99±0.03 AU) and protein levels (0.58±0.05 AU versus 0.34±0.04 AU) and enhanced NADPH oxidase activity (1998±181 counts/s versus 1322±112 counts/s). No differences in these parameters were observed among genotypes in normotensive cells. Transfection experiments on vascular smooth muscle cells showed that the A-to-G substitution of this polymorphism produced an increased reporter gene expression in hypertensive cells. Nitric oxide production, as assessed by measurement of serum nitric oxide metabolites, was lower in GG hypertensive subjects than in AA/AG hypertensive subjects. In conclusion, these results suggest that hypertensive subjects carrying the GG genotype of the p22phox −930A/G polymorphism are highly exposed to NADPH oxidase-mediated oxidative stress.

The C242T CYBA polymorphism of NADPH oxidase is associated with essential hypertension.

Objective Oxidative stress is implicated in hypertension. The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are the main source of superoxide in phagocytic and vascular cells. The C242T polymorphism of CYBA, the human gene that encodes p22phox, has been found to be functionally associated with vascular NADPH oxidase activity in atherosclerotic patients. We investigated the association of the C242T polymorphism with hypertension and its potential impact on NADPH oxidase activity. We also analysed the interaction of C242T polymorphism with the –930A/G CYBA variant. Design Case–control study in a random sample of 623 subjects (326 hypertensive patients and 297 normotensive controls) from the general population. Methods CYBA polymorphisms were determined by restriction fragment length polymorphism (RFLP) or allelic discrimination. NADPH oxidase activity and p22phox expression were quantified in phagocytic cells by chemiluminescence and by northern and western blots, respectively. Results The prevalence of the CC genotype and the C allele frequency were significantly higher (P < 0.05) in hypertensives than in normotensives. CC genotype remained associated with hypertension after adjusting for potential confounders in a logistic regression analysis. Increased phagocytic NADPH oxidase activity was observed in CC hypertensives compared with CT and TT hypertensives (P < 0.05). Enhanced plasma levels of von Willebrand factor were found in CC hypertensives compared with TT hypertensives (P < 0.05). The C242T polymorphism was not in linkage disequilibrium with the −930A/G CYBA promoter variation, which also associates with hypertension. Conclusion The C242T CYBA polymorphism is associated with essential hypertension. Furthermore, hypertensives carrying the CC genotype of this polymorphism exhibit features of NADPH oxidase-mediated oxidative stress and endothelial damage.

NADPH oxidase CYBA polymorphisms, oxidative stress and cardiovascular diseases

Oxidative stress plays a key role in the pathophysiology of several major cardiovascular diseases, including atherosclerosis, hypertension, heart failure, stroke and diabetes. ROS (reactive oxygen species) affect multiple tissues either directly or through NO depletion. ROS induce cardiovascular dysfunction by modulating cell contraction/dilation, migration, growth/apoptosis and extracellular matrix protein turnover, which contribute to vascular and cardiac remodelling. Of the several sources of ROS within the cardiovascular system, a family of multisubunit NADPH oxidases appears to be a predominant contributor of superoxide anion. Recent findings suggest a significant role of the genetic background in NADPH oxidase regulation. Common genetic polymorphisms within the promoter and exonic sequences of CYBA, the gene that encodes the p22(phox) subunit of NADPH oxidase, have been characterized in the context of cardiovascular diseases. This review aims to present the current state of research into these polymorphisms in their relationship to cardiovascular diseases.

Phagocytic NADPH Oxidase-Dependent Superoxide Production Stimulates Matrix Metalloproteinase-9: Implications for Human Atherosclerosis

Objective—Data suggest that matrix metalloproteinase-9 (MMP-9) has a role in atherosclerosis. The phagocytic NADPH oxidase has been also associated with atherosclerosis. This study aimed to investigate the association between phagocytic NADPH oxidase and MMP-9 in human atherosclerosis. Methods and Results—In vitro experiments performed in human monocytes showed that NADPH oxidase activation enhanced MMP-9 secretion and activity, determined by enzyme-linked immunosorbent assay and zymography, respectively. Immunohistochemical study showed that phagocytic NADPH oxidase localized with MMP-9 in endarterectomies from patients with carotid stenosis. In addition, a positive relationship (P<0.001) was found between phagocytic NADPH oxidase-dependent superoxide production determined with lucigenin and plasma MMP-9 levels in 188 asymptomatic subjects free of overt clinical atherosclerosis. In multivariate analysis, this association remained significant after adjustment for cardiovascular risk factors. Interestingly, subjects in the upper quartile of superoxide production exhibited the highest values of MMP-9, oxidized low-density lipoprotein, nitrotyrosine, carotid intima media thickness, and an increased presence of carotid plaques. Conclusions—Enhanced NADPH oxidase-dependent ·O2− production stimulates MMP-9 in monocytes and this relationship may be relevant in the atherosclerotic process. Moreover, MMP-9 emerges as an important mediator of the phagocytic NADPH oxidase-dependent oxidative stress in atherosclerosis.

Ultrasonic Backscatter and Serum Marker of Cardiac Fibrosis in Hypertensives

Elevations in the serum concentration of the carboxy-terminal propeptide of procollagen type I (PIP) >127 &mgr;g/L have been found to predict severe myocardial fibrosis in hypertensive patients. This study was designed to assess whether ultrasonic reflectivity, evaluated by a real-time integrated backscatter analysis, was related to the severity of myocardial fibrosis as estimated by serum PIP. Thirty-four subjects were included in the study. Serum PIP was measured by specific radio immunoanalysis. Backscatter cyclic variation and maximal intensity were measured in 6 regions throughout the left ventricle. The subjects were divided into 3 groups: 14 normotensives with PIP <127 &mgr;g/L (group 1), 12 hypertensives with PIP <127 &mgr;g/L (group 2), and 8 hypertensives with PIP >127 &mgr;g/L (group 3). The highest cyclic variation was found in group 1 and the lowest in group 3 (5.78±0.25 versus 4.70±0.33 dB, P <0.05), with intermediate values in group 2 (5.10±0.27 dB). No differences in maximal intensity were found among the 3 groups of subjects. Using receiver operating characteristics curves, we observed that a cutoff of 2.90 dB for cyclic variation measured in the apex provided 75% sensitivity and 63% specificity for predicting PIP >127 &mgr;g/L in hypertensives, with a relative risk of 2.50 (95% CI, 0.72 to 34.70). These results show an association between diminished cyclic variation of backscatter and increased serum concentration of PIP in hypertension. Thus, the combination of these 2 parameters may be useful for the diagnosis of severe myocardial fibrosis associated with hypertension.

NADPH Oxidase–Dependent Superoxide Production Is Associated With Carotid Intima-Media Thickness in Subjects Free of Clinical Atherosclerotic Disease

Objective—Oxidative stress plays a critical role in the pathogenesis of atherosclerosis. The NADPH oxidase constitutes the main source of superoxide in phagocytic and vascular cells. This study aimed to investigate the levels of NADPH oxidase–mediated superoxide production in phagocytic cells and the association between phagocytic superoxide production and carotid intima-media thickness (IMT), a surrogate marker of asymptomatic atherosclerosis. Methods and Results—NADPH oxidase–mediated superoxide production was determined by a chemiluminescence assay using lucigenin and associated with IMT for 184 asymptomatic subjects free of overt clinical atherosclerotic disease. Compared with individuals in the lowest tertile of superoxide production, those in the upper tertile (>20 counts/sec) showed significantly higher IMT (P<0.05). In correlation analysis, a positive relationship was found between superoxide production and carotid IMT. Superoxide production also correlated positively (P<0.05) with body mass index (BMI). In multivariate analysis, the association of superoxide production with carotid IMT remained significant after adjustment for age, sex, systolic blood pressure, BMI, triglycerides, glucose, and smoking. Conclusions—In a population sample of adults without clinically overt atherosclerotic disease, increased NADPH oxidase activity was associated with enhanced carotid IMT, suggesting a relationship between phagocytic NADPH oxidase–mediated oxidative stress and the development of atherosclerosis.

Is plasma cardiotrophin-1 a marker of hypertensive heart disease?

Objective This study was designed to investigate whether plasma concentration of cardiotrophin-1 (CT-1), a cytokine that induces cardiomyocyte hypertrophy and stimulates cardiac fibroblasts, is related to hypertensive heart disease, as defined by the presence of echocardiographically assessed left ventricular hypertrophy (LVH). Methods The study was performed in 31 normotensive subjects and 111 patients with never-treated essential hypertension (54 without LVH and 57 with LVH). Causes of LVH other than hypertension were excluded after a complete medical workup. A novel enzyme-linked immunosorbent assay was developed to measure plasma CT-1. Results Plasma CT-1 was increased (P < 0.001) in hypertensives compared with normotensives. The value of CT-1 was higher (P < 0.001) in hypertensives with LVH than in hypertensives without LVH. Some 31% of patients without LVH exhibited values of CT-1 above the upper normal limit in normotensives. A direct correlation was found between CT-1 and left ventricular mass index (r = 0.319, P < 0.001) in all subjects. Receiver operating characteristic curves showed that a cutoff of 39 fmol/ml for CT-1 provided 75% specificity and 70% sensitivity for predicting LVH with a relative risk of 6.21 (95% confidence interval, 2.95 to 13.09). Conclusions These results show an association between LVH and the plasma concentration of CT-1 in essential hypertension. Although preliminary, these findings suggest that the determination of CT-1 may be an easy and reliable method for the initial screening and diagnosis of hypertensive heart disease.