Ana Fortuño

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.

Oxidative stress and vascular remodelling

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.

Adipose tissue as an endocrine organ: role of leptin and adiponectin in the pathogenesis of cardiovascular diseases

Obesity, the most common nutritional disorder in industrial countries, is associated with increased cardiovascular mortality and morbidity. Nevertheless, the molecular basis linking obesity with cardiovascular disturbances have not yet been fully clarified. Recent advances in the biology of adipose tissue indicate that it is not simply an energy storage organ, but also a secretory organ, producing a variety of bioactive substances, including leptin and adiponectin, that may influence the function as well as the structural integrity of the cardiovascular system. Leptin, besides being a satiety signal for the central nervous system and to be related to insulin and glucose metabolism, may also play an important role in regulating vascular tone because of the widespread distribution of functional receptors in the vascular cells. On the other hand, the more recently discovered protein, adiponectin, seems to play a protective role in experimental models of vascular injury, in probable relation to its ability to suppress the attachment of monocytes to endothelial cells, which is an early event in the atherosclerotic process. There is already considerable evidence linking altered production of some adipocyte hormones with the cardiovascular complications of obesity. Therefore, the knowledge of alterations in the endocrine function of adipose tissue may help to further understand the high cardiovascular risk associated with obesity.ResumenLa obesidad es la más común de las enfermedades nutricionales de los países industrializados y está asociada con un aumento de la mortalidad y morbilidad cardiovascular. Sin embargo, las bases moleculares de esta asociación aún no se han clarificado. Recientes avances en la biología del tejido adiposo han permitido demostrar que además de almacenar energía, es también un órgano de secreción capaz de producir una amplia variedad de sustancias bioactivas entre las que se incluyen la leptina y la adiponectina. Ambas hormonas pueden influir tanto en la función como en la integridad estructural del sistema cardiovascular. La leptina, además de ser una señal de saciedad para el sistema nervioso central y estar relacionada con el metabolismo de la glucosa y de la insulina, desempeña también un papel en la regulación del tono vascular, como consecuencia de la amplia distribución de sus receptores funcionales en las células vasculares. Por otra parte, la adiponectina, proteína descrita más recientemente, parece jugar un papel protector en los modelos experimentales de daño vascular, en parte, porque es capaz de suprimir la unión de los monocitos a las células endoteliales, que es un paso fundamental en el proceso aterosclerótico. Existen suficientes evidencias que relacionan alteraciones de la producción de hormonas adipocitarias con las complicaciones cardiovasculares de la obesidad. En consecuencia, el avance en el conocimiento de las alteraciones en la función endocrina del tejido adiposo podría ayudar a entender el riesgo cardiovascular asociado a la obesidad.

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.

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.

Polymorphisms and Promoter Overactivity of the p22phox Gene in Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats

Abstract — 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 (·O2−). 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 ≈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, &ggr;-interferon, and nuclear factor-&kgr;B. 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.

Mechanisms of increased susceptibility to angiotensin II-induced apoptosis in ventricular cardiomyocytes of spontaneously hypertensive rats.

Previous findings have shown that hypotensive doses of losartan prevent the excess of apoptosis present in the hypertrophied left ventricle of adult spontaneously hypertensive rats (SHR). This study was designed to determine whether angiotensin II facilitates apoptosis in cardiomyocytes of adult SHR. Primary cultures of ventricular cardiomyocytes from 30-week-old normotensive Wistar-Kyoto rats (WKY) and SHR with left ventricular hypertrophy were exposed to 10−9 mol/L angiotensin II for 24 hours. Apoptotic cells were assessed by terminal deoxynucleotidyl transferase assay and confirmed by Annexin V detection. The expression of Bax-&agr;, Bcl-2, p53 , and caspase-3 proteins was assessed by Western blot assays. The expression of BAX gene was assessed by Northern blot. Angiotensin II increased (P <0.01) cardiomyocyte apoptosis, and this effect was higher (P <0.001) in SHR cells than in WKY cells. Whereas losartan (10−7 mol/L) blocked the apoptotic effect of the octapeptide in cells from the two strains of rats, PD123319 (10−7 mol/L) inhibited angiotensin II–mediated apoptosis only in SHR cells. Angiotensin II stimulated (P <0.01) Bax-&agr; protein, and this effect was higher (P <0.01) in SHR cells than in WKY cells. Angiotensin II did not modify Bcl-2, p53, and BAX mRNA in cells from the two strains of rats. Angiotensin II induced a similar increase (P <0.05) in the ratio caspase-3/procaspase-3 (an index of caspase-3 activation) in cardiomyocytes from the two strains of rats. The present in vitro results indicate that SHR cardiomyocytes exhibit enhanced susceptibility to angiotensin II–induced apoptosis. Ligand binding to angiotensin II type 1 and type 2 receptors leading to changes in posttranscriptional processing of Bax-&agr; and accumulation of this proapoptotic protein may be involved in the abnormal response of SHR cardiomyocytes. These data support a role for angiotensin II in apoptosis observed in the left ventricle of these rats.