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

Increased phagocytic nicotinamide adenine dinucleotide phosphate oxidase–dependent superoxide production in patients with early chronic kidney disease

Background. Oxidative stress has been implicated in the pathogenesis of atherosclerosis that develops in patients with advanced chronic kidney disease (CKD). This study was designed to investigate whether a relationship exists between phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase–dependent superoxide anion (•O2 −) production and subclinical atherosclerosis in patients with early CKD. Methods. Superoxide production was assayed by chemiluminescence under baseline and stimulated conditions on mononuclear cells obtained from asymptomatic patients with stage 1 to 2 CKD (N = 22) and healthy controls (N = 21). Ultrasonographic determination of carotid intima-media thickness (IMT) was used to assess the presence of atherosclerosis. Results. Although there were no differences in baseline •O2 − production between controls and patients, the •O2 − production in phorbol myristate acetate–stimulated mononuclear cells was increased (P < 0.05) in patients compared with controls. The phorbol myristate acetate–induced •O2 − production was completely abolished by apocynin, a specific inhibitor of NADPH oxidase. A direct correlation (r = 0.441, P < 0.05) was found between plasma insulin levels and NADPH oxidase–mediated •O2 − production in patients. Carotid IMT was higher (P < 0.005) in patients than in controls. CarotidIMTvalues above the upper normal limit in controls were found in 70% and 40% of patients with increased or normal NADPH oxidase–mediated •O2 − production, respectively. Conclusion. Generation of •O2 − that is mainly dependent on NADPH oxidase is abnormally enhanced in patients with early CKD. It is suggested that this alteration could be related to the development of subclinical atherosclerosis in these patients

Polymorphisms and promoter overactivity of the p22(phox) gene in vascular smooth muscle cells from spontaneously hypertensive rats

In a previous study, we found that the p22(phox) subunit of the NADH/NADPH oxidase is overexpressed in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHRs) with enhanced vascular production of superoxide anion ((.)O(2)(-)). Thus, we have investigated whether changes in the sequence or activity of the promoter region of p22(phox) gene are present in SHRs. To carry out this analysis, first of all, we characterized the rat gene structure and promoter region for the p22(phox) subunit. The p22(phox) gene spans approximately 10 kb and contains 6 exons and 5 introns. Primer extension analysis indicated the transcriptional start site 100 bp upstream from the translational start site. The immediate promoter region of the p22(phox) gene does not contain a TATA box, but there are a CCAC box and putative recognition sites for nuclear factors, such as SP1, gamma-interferon, and nuclear factor-kappaB. Using reporter-gene transfection analysis, we found that this promoter was functional in VSMCs. Furthermore, we observed that p22(phox) promoter activity was significantly higher in VSMCs from SHRs than from normotensive Wistar-Kyoto rats. In addition, we found that there were 5 polymorphisms in the sequence of p22(phox) promoter between Wistar-Kyoto rats and SHRs and that they were functional. The results obtained in this study provide a tool to explore the mechanisms that regulate the expression of p22(phox) gene in rat VSMCs. Furthermore, our findings show that changes in the sequence of p22(phox) gene promoter and in the degree of activation of VSMCs are responsible for upregulated expression of p22(phox) in SHRs

Cardiac Fibrosis in heart failure: Focus on non-invasive diagnosis and emerging therapeutic strategies

Heart failure is a leading cause of mortality and hospitalization worldwide. Cardiac fibrosis, resulting from the excessive deposition of collagen fibers, is a common feature across the spectrum of conditions converging in heart failure. Eventually, either reparative or reactive in nature, in the long-term cardiac fibrosis contributes to heart failure development and progression and is associated with poor clinical outcomes. Despite this, specific cardiac antifibrotic therapies are lacking, making cardiac fibrosis an urgent unmet medical need. In this context, a better patient phenotyping is needed to characterize the heterogenous features of cardiac fibrosis to advance toward its personalized management. In this review, we will describe the different phenotypes associated with cardiac fibrosis in heart failure and we will focus on the potential usefulness of imaging techniques and circulating biomarkers for the non-invasive characterization and phenotyping of this condition and for tracking its clinical impact. We will also recapitulate the cardiac antifibrotic effects of existing heart failure and non-heart failure drugs and we will discuss potential strategies under preclinical development targeting the activation of cardiac fibroblasts at different levels, as well as targeting additional extracardiac processes

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

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

The p22(phox) subunit is an essential protein in the activation of NAD(P)H oxidase. Here we report the preliminary characterisation of the human p22(phox) gene promoter. The p22(phox) promoter contains TATA and CCAC boxes and Sp1, gamma-interferon and nuclear factor kappaB sites. We screened for mutations in the p22(phox) 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 -930(A/G) polymorphism in the p22(phox) promoter may be a novel genetic marker associated with hypertension

Association of increased phagocytic NADPH oxidasedependent 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

Oxidative Stress in Arterial Hypertension: Role of NAD(P)H Oxidase

Increased vascular reactive oxygen species production, especially superoxide anion, contributes significantly in the functional and structural alterations present in hypertension. An enhanced superoxide production causes a diminished NO bioavailability by an oxidative reaction that inactivates NO. Exaggerated superoxide levels and a low NO bioavailability lead to endothelial dysfunction and hypertrophy of vascular cells. It has been shown that the enzyme NAD(P)H oxidase plays a major role as the most important source of superoxide anion in vascular cells. Several experimental observations have shown an enhanced superoxide generation as a result of the activation of vascular NAD(P)H oxidase in hypertension. Although this enzyme responds to stimuli such as vasoactive factors, growth factors, and cytokines, some recent data suggest the existence of a genetic background modulating the expression of its different components. New polymorphisms have been identified in the promoter of the p22(phox) gene, an essential subunit of NAD(P)H oxidase, influencing the activity of this enzyme. Genetic investigations of these polymorphisms will provide novel markers for determination of genetic susceptibility to oxidative stress in hypertension

The A640G CYBA polymorphism associates with subclinical atherosclerosis in diabetes

Oxidative stress is implicated in diabetes. The NADPH oxidases are the main source of superoxide in phagocytic and vascular cells, and p22phox is a key subunit. Genetic variants of CYBA, the human p22phox gene, associate with cardiovascular disease. We investigated the association of the A640G polymorphism with diabetes and its impact on phagocytic NADPH oxidase-dependent superoxide production and subclinical atherosclerosis. We studied 1212 subjects in which clinical parameters including carotid intima-media thickness (cIMT) were assessed. The A640G polymorphism was genotyped by TaqMan probes. In 496 subjects, the NADPH oxidase-dependent superoxide production in peripheral blood mononuclear cells was assessed by chemiluminescence. The GG genotype prevalence was significantly higher in type 2 diabetic patients than in non-diabetic subjects. Peripheral blood mononuclear cells from diabetic GG patients presented higher NADPH oxidase-dependent superoxide production than those of diabetic AA/AG patients. Within the diabetic group, GG patients presented higher cIMT levels than AA/AG patients. The A640G CYBA polymorphism may be a marker of oxidative stress risk and may be indicative of subclinical atherosclerosis in type 2 diabetes

Vascular NADH/NADPH oxidase is involved in enhanced superoxide production in spontaneously hypertensive rats

This study was designed to test the hypothesis that stimulation of nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase is involved in increased vascular superoxide anion (*O(2)(-)) production in spontaneously hypertensive rats (SHR). The study was performed in 16-week-old and 30-week-old normotensive Wistar-Kyoto rats (WKY(16) and WKY(30), respectively) and in 16-week-old and 30-week-old SHR (SHR(16) and SHR(30), respectively). In addition, 16-week-old SHR were treated with oral irbesartan (average dose 20 mg/kg per day) for 14 weeks (SHR(30)-I). Aortic NADH/NADPH oxidase activity was determined by use of chemiluminescence with lucigenin. The expression of p22phox messenger RNA was assessed by competitive reverse transcription-polymerase chain reaction. Vascular responses to acetylcholine were determined by isometric tension studies. Aortic wall structure was studied, determining the media thickness and the cross-sectional area by morphometric analysis. Whereas systolic blood pressure was significantly increased in the 2 groups of hypertensive animals compared with their normotensive controls, no differences were observed in systolic blood pressure between SHR(30) and SHR(16). No other differences in the parameters measured were found between WKY(16) and SHR(16). In SHR(30) compared with WKY(30), we found significantly greater p22phox mRNA level, NADH/NADPH-driven *O(2)(-) production, media thickness, and cross-sectional area and an impaired vasodilation in response to acetylcholine. Treated SHR had similar NADH/NADPH oxidase activity and p22phox expression as the WKY(30) group. The vascular functional and morphological parameters were improved in SHR(30)-I. These findings suggest that an association exists between p22phox gene overexpression and NADH/NADPH overactivity in the aortas of adult SHR. Enhanced NADH/NADPH oxidase-dependent *O(2)(-) production may contribute to endothelial dysfunction and vascular hypertrophy in this genetic model of hypertension