Abstract-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 (⅐O 2 Ϫ ) 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 ⅐O 2 Ϫ 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 ⅐O 2 Ϫ production may contribute to endothelial dysfunction and vascular hypertrophy in this genetic model of hypertension.

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.

Myocardial fibrosis impairs cardiac function, in addition to facilitating arrhythmias and ischemia, and thus influences the evolution and outcome of cardiac diseases. Its assessment is therefore clinically relevant. Although tissue biopsy is the gold standard for the diagnosis of myocardial fibrosis, a number of circulating biomarkers have been proposed for the noninvasive assessment of this lesion. A review of the published clinical data available on these biomarkers shows that most of them lack proof that they actually reflect the myocardial accumulation of fibrous tissue. In this "call to action" article, we propose that this absence of proof may lead to misinterpretations when considering the incremental value provided by the biomarkers with respect to traditional diagnostic tools in the clinical handling of patients. We thus argue that strategies are needed to more strictly validate whether a given circulating biomarker actually reflects histologically proven myocardial fibrosis before it is applied clinically.

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Excessive myocardial CCL is associated with HHF in hypertensive patients with HF. In this population, the serum CITP:MMP-1 ratio identifies patients with increased CCL and high risk of HHF.

The p22phox 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, Q Q-interferon and nuclear factor U UB sites. We screened for mutations in the p22 phox promoter and identi¢ed a new polymorphism, localised at position 3 3930 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 3 3930 A=G polymorphism in the p22 phox promoter may be a novel genetic marker associated with hypertension.

Oxidative stress plays an important role in the pathophysiology of vascular diseases. Reactive oxygen species, especially superoxide anion and hydrogen peroxide, are important signalling molecules in cardiovascular cells. Enhanced superoxide production increases nitric oxide inactivation and leads to an accumulation of peroxynitrites and hydrogen peroxide. Reactive oxygen species participate in growth, apoptosis and migration of vascular smooth muscle cells, in the modulation of endothelial function, including endothelium-dependent relaxation and expression of proinflammatory phenotype, and in the modification of the extracellular matrix. All these events play important roles in vascular diseases such as hypertension, suggesting that the sources of reactive oxygen species and the signalling pathways that they modify may represent important therapeutic targets. Potential sources of vascular superoxide production include NADPH-dependent oxidases, xanthine oxidases, lipoxygenases, mitochondrial oxidases and nitric oxide synthases. Studies performed during the last decade have shown that NADPH oxidase is the most important source of superoxide anion in phagocytic and vascular cells. Evidence from experimental animal and human studies suggests a significant role of NADPH oxidase activation in the vascular remodelling and endothelial dysfunction found in cardiovascular diseases.

The term oxidative stress refers to a situation in which cells are exposed to excessive levels of either molecular oxygen or chemical derivatives of oxygen (ie, reactive oxygen species). Three enzyme systems produce reactive oxygen species in the vascular wall: NADH/NADPH oxidase, xanthine oxidoreductase, and endothelial nitric oxide synthase. Among vascular reactive oxygen species superoxide anion plays a critical role in vascular biology because it is the source for many other reactive oxygen species and various vascular cell functions. It is currently thought that increases in oxidant stress, namely excessive production of superoxide anion, are involved in the pathophysiology of endothelial dysfunction that accompanies a number of cardiovascular risk factors including hypercholesterolemia, hypertension and cigarette smoking. On the other hand, vascular oxidant stress plays a pivotal role in the evolution of clinical conditions such as atherosclerosis, diabetes and heart failure.