NADPH Oxidase Overactivity Underlies Telomere Shortening in Human Atherosclerosis.

Telomere shortening and oxidative stress are involved in the pathogenesis of atherosclerosis. Different studies have shown that phagocytic NADPH oxidase is associated with this disease. This study aimed to investigate the association between phagocytic NADPH oxidase and telomere shortening in human atherosclerosis. To assess this potential association, telomere length and phagocytic NADPH oxidase activity were determined by PCR and chemiluminescence, respectively, in a population of asymptomatic subjects free of overt clinical atherosclerosis. We also measured serum 8-hydroxy-2-deoxyguanosine (8-OHdG) levels (an index of oxidative stress) and carotid intima-media thickness (IMT), a surrogate marker of atherosclerosis. After adjusting them for age and sex, telomere length inversely correlated (p < 0.05) with NADPH oxidase-mediated superoxide production, with 8-OHdG values, and with carotid IMT. Interestingly, the asymptomatic subjects with plaques have a lower telomere length (p < 0.05), and higher values of plasma 8-OHdG and superoxide production (p < 0.05). These data were confirmed in a second population in which patients with coronary artery disease showed lower telomere length and higher 8-OHdG and superoxide production than the asymptomatic subjects. In both studies, NADPH oxidase-dependent superoxide production in phagocytic cells was only due to the specific expression of the Nox2 isoform. In conclusion, these findings suggest that phagocytic NADPH oxidase may be involved in oxidative stress-mediated telomere shortening, and that this axis may be critically involved in human atherosclerosis.

Leptin Induces Oxidative Stress Through Activation of NADPH Oxidase in Renal Tubular Cells: Antioxidant Effect of L-Carnitine.

Leptin is a protein involved in the regulation of food intake and in the immune and inflammatory responses, among other functions. Evidences demonstrate that obesity is directly associated with high levels of leptin, suggesting that leptin may directly link obesity with the elevated cardiovascular and renal risk associated with increased body weight. Adverse effects of leptin include oxidative stress mediated by activation of NADPH oxidase. The aim of this study was to evaluate the effect of L-carnitine (LC) in rat renal epithelial cells (NRK-52E) exposed to leptin in order to generate a state of oxidative stress characteristic of obesity. Leptin increased superoxide anion (O2 (•) -) generation from NADPH oxidase (via PI3 K/Akt pathway), NOX2 expression and nitrotyrosine levels. On the other hand, NOX4 expression and hydrogen peroxide (H2 O2 ) levels diminished after leptin treatment. Furthermore, the expression of antioxidant enzymes, catalase, and superoxide dismutase, was altered by leptin, and an increase in the mRNA expression of pro-inflammatory factors was also found in leptin-treated cells. LC restored all changes induced by leptin to those levels found in untreated cells. In conclusion, stimulation of NRK-52E cells with leptin induced a state of oxidative stress and inflammation that could be reversed by preincubation with LC. Interestingly, LC induced an upregulation of NOX4 and restored the release of its product, hydrogen peroxide, which suggests a protective role of NOX4 against leptin-induced renal damage. J. Cell. Biochem. 117: 2281-2288, 2016. © 2016 Wiley Periodicals, Inc.

Mercury induces proliferation and reduces cell size in vascular smooth muscle cells through MAPK, oxidative stress and cyclooxygenase-2 pathways.

Mercury exposure is known to increase cardiovascular risk but the underlying cellular mechanisms remain undetermined. We analyzed whether chronic exposure to HgCl2 affects vascular structure and the functional properties of vascular smooth muscle cells (VSMC) through oxidative stress/cyclooxygenase-2 dependent pathways. Mesenteric resistance arteries and aortas from Wistar rats treated with HgCl2 (first dose 4.6mgkg(-1), subsequent doses 0.07mgkg(-1)day(-1), 30days) and cultured aortic VSMC stimulated with HgCl2 (0.05-5µg/ml) were used. Treatment of rats with HgCl2 decreased wall thickness of the resistance and conductance vasculature, increased the number of SMC within the media and decreased SMC nucleus size. In VSMCs, exposure to HgCl2: 1) induced a proliferative response and a reduction in cell size; 2) increased superoxide anion production, NADPH oxidase activity, gene and/or protein levels of the NADPH oxidase subunit NOX-1, the EC- and Mn-superoxide dismutases and cyclooxygenase-2 (COX-2); 3) induced activation of ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized the proliferative response and the altered cell size induced by HgCl2. Blockade of ERK1/2 and p38 signaling pathways abolished the HgCl2-induced Nox1 and COX-2 expression and normalized the alterations induced by mercury in cell proliferation and size. In conclusion, long exposure of VSMC to low doses of mercury activates MAPK signaling pathways that result in activation of inflammatory proteins such as NADPH oxidase and COX-2 that in turn induce proliferation of VSMC and changes in cell size. These findings offer further evidence that mercury might be considered an environmental risk factor for cardiovascular disease.

Decreased Nox4 levels in the myocardium of patients with aortic valve stenosis.

The NADPH oxidases are a key family of ROS (reactive oxygen species)-producing enzymes which may differentially contribute to cardiac pathophysiology. Animal studies show uncertain results regarding the regulation of cardiac Nox4 by pressure overload and no data are available on human myocardial Nox4. In the present study, we evaluated Nox4 expression and its relationship with myocardial remodelling and LV (left ventricular) function in patients with severe AS (aortic valve stenosis). Endomyocardial biopsies from 34 patients with AS were obtained during aortic valve replacement surgery. LV morphology and function were assessed by echocardiography. Myocardial samples from subjects deceased of non-CVDs (cardiovascular diseases) were analysed as controls. Nox4 localization was evaluated by immunohistochemistry and quantified by Western blot. Myocardial capillary density, fibrosis and cardiomyocyte dimensions and apoptosis were assessed histologically to evaluate myocardial remodelling. Nox4 was present in samples from all subjects and expressed in cardiomyocytes, VSMCs (vascular smooth muscle cells), endothelium and fibroblasts. Nox4 levels were reduced 5-fold in AS patients compared with controls (P<0.01). Nox4 levels directly correlated with cardiomyocyte cross-sectional area (r=0.299, P<0.05) and diameter (r=0.406, P<0.05) and capillary density (r=0.389, P<0.05), and inversely with cardiomyocyte apoptosis (r=-0.316, P<0.05) in AS patients. In addition, Nox4 levels correlated with echocardiographic parameters (LV ejection fraction: r=0.353, P<0.05; midwall fractional shortening: r=0.355, P<0.05; deceleration time: r=-0.345, P<0.05) in AS patients. Nox4 is expressed in human myocardium and reduced in AS patients. The observed associations of Nox4 with cardiomyocyte parameters and capillary density in AS patients suggest a potential role of Nox4 deficiency in the myocardial remodelling present in the human pressure-overloaded heart.

Leptin inhibits the proliferation of vascular smooth muscle cells induced by angiotensin II through nitric oxide-dependent mechanisms.

OBJECTIVE: This study was designed to investigate whether leptin modifies angiotensin (Ang) II-induced proliferation of aortic vascular smooth muscle cells (VSMCs) from 10-week-old male Wistar and spontaneously hypertensive rats (SHR), and the possible role of nitric oxide (NO). METHODS: NO and NO synthase (NOS) activity were assessed by the Griess and (3)H-arginine/citrulline conversion assays, respectively. Inducible NOS (iNOS) and NADPH oxidase subutnit Nox2 expression was determined by Western-blot. The proliferative responses to Ang II were evaluated through enzymatic methods. RESULTS: Leptin inhibited the Ang II-induced proliferative response of VSMCs from control rats. This inhibitory effect of leptin was abolished by NOS inhibitor, NMMA, and iNOS selective inhibitor, L-NIL, and was not observed in leptin receptor-deficient fa/fa rats. SHR showed increased serum leptin concentrations and lipid peroxidation. Despite a similar leptin-induced iNOS up-regulation, VSMCs from SHR showed an impaired NOS activity and NO production induced by leptin, and an increased basal Nox2 expression. The inhibitory effect of leptin on Ang II-induced VSMC proliferation was attenuated. CONCLUSION: Leptin blocks the proliferative response to Ang II through NO-dependent mechanisms. The attenuation of this inhibitory effect of leptin in spontaneous hypertension appears to be due to a reduced NO bioavailability in VSMCs.

The angiotensin-converting enzyme insertion/deletion polymorphism is associated with phagocytic NADPH oxidase-dependent superoxide generation: potential implication in hypertension.

The objective of the present study was to analyse the influence of the ACE (angiotensin-converting enzyme) gene I/D (insertion/deletion) polymorphism on NADPH oxidase-dependent O(2)(*-) (superoxide radical) production, and to investigate the clinical implication of this association in hypertensive subjects. A case-control study was performed in a random sample of the general population composed of 189 normotensive subjects and 223 hypertensive subjects. The ACE polymorphism was determined by PCR. NADPH oxidase-dependent O(2)(*-) production was quantified in phagocytic cells by chemiluminescence. MMP-9 (matrix metalloproteinase-9), a marker of atherosclerosis previously reported to be associated with NADPH oxidase overactivity, was quantified by ELISA in plasma samples. The distribution of genotypes was in Hardy-Weinberg equilibrium. The I/D polymorphism was not associated with hypertension. NADPH oxidase-dependent O(2)(*-) production was significantly higher in D/D (deletion/deletion) than in I/I (insertion/insertion) and I/D, both in normotensive and hypertensive subjects. Interestingly, plasma levels of angiotensin II were significantly higher in D/D than in I/I and I/D, both in normotensive and hypertensive subjects. Plasma levels of MMP-9 and systolic blood pressure values were significantly higher in D/D than in I/I and I/D hypertensive subjects, whereas no differences were found among genotypes in normotensive subjects. Interestingly, NADPH oxidase-dependent O(2)(*-) production positively associated with plasma MMP-9 levels in hypertensive subjects, which remained significant after adjustment for age and gender. In conclusion, in the present study we have reported for the first time an association of the D/D genotype of the ACE I/D polymorphism with phagocytic NADPH oxidase-mediated O(2)(*-) overproduction. Within the group of hypertensive patients, D/D cases also associated with increased blood pressure values and with enhanced plasma levels of MMP-9.

Molecular mechanisms of atherosclerosis in metabolic syndrome: role of reduced IRS2-dependent signaling.

OBJECTIVE: The mechanisms underlying accelerated atherosclerosis in metabolic syndrome (MetS) patients remain poorly defined. In the mouse, complete disruption of insulin receptor substrate-2 (Irs2) causes insulin resistance, MetS-like manifestations, and accelerates atherosclerosis. Here, we performed human, mouse, and cell culture studies to gain insight into the contribution of defective Irs2 signaling to MetS-associated alterations. METHODS AND RESULTS: In circulating leukocytes from insulin-resistant MetS patients, Irs2 and Akt2 mRNA levels inversely correlate with plasma insulin levels and HOMA index and are reduced compared to insulin-sensitive MetS patients. Notably, a moderate reduction in Irs2 expression in fat-fed apolipoprotein E-null mice lacking one allele of Irs2 (apoE(-/-)Irs2(+/-)) accelerates atherosclerosis compared to apoE-null controls, without affecting plaque composition. Partial Irs2 inactivation also increases CD36 and SRA scavenger receptor expression and modified LDL uptake in macrophages, diminishes Akt2 and Ras expression in aorta, and enhances expression of the proatherogenic cytokine MCP1 in aorta and primary vascular smooth muscle cells (VSMCs) and macrophages. Inhibition of AKT or ERK1/2, a downstream target of RAS, upregulates Mcp1 in VSMCs. CONCLUSIONS: Enhanced levels of MCP1 resulting from reduced IRS2 expression and accompanying defects in AKT2 and Ras/ERK1/2 signaling pathways may contribute to accelerated atherosclerosis in MetS states.

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.

Vasodilator effect of ghrelin in the rat aorta.

BACKGROUND AND OBJECTIVE: Ghrelin is a hormone mainly produced by the stomach and has been identified as the natural ligand of the growth hormone secretagogue receptor (GHS-R). In addition to its orexigenic effect, ghrelin modulates homeostasis of the cardiovascular system. The aim of this study was to analyze the effect of ghrelin on the vascular reactivity of the aorta. MATERIAL AND METHODS: This study was performed in the thoracic aorta of 10-12- week-old male Wistar rats. Passive wall tension and contractile responses to angiotensin II (Ang II) (10(-7) mol/l) in the presence of ghrelin (10(-8) mol/l) were evaluated using the organ bath system. The presence of the functional ghrelin receptor, GHS-R 1a, in vascular smooth muscle cells (VSMCs) of the aorta was determined by Western blot. RESULTS: VSMCs of the vascular wall of the aorta expressed the GHS-R 1a protein. Incubation of the intact aorta with ghrelin (10(-8) mol/l) induced a reduction (p<0.01) in passive wall tension. In endotheliumdenuded aorta, ghrelin did not modify basal vasomotor tone. However, incubation of endothelium-denuded aorta with ghrelin inhibited (p<0.01) Ang II (10(-7) mol/l)-induced vasoconstriction. CONCLUSIONS: Ghrelin exerts an endothelium-dependent and -independent depressor action on aortic vascular reactivity. These findings provide additional information on the physiological effect of ghrelin on arterial pressure regulation.

Sleep-Disordered Breathing Is Independently Associated With Increased Aggressiveness of Cutaneous Melanoma: A Multicenter Observational Study in 443 Patients.

BACKGROUND: Sleep-disordered breathing (SDB) has been associated with a greater incidence and mortality of cancer, although such findings are inconsistent. However, no large studies are currently available to investigate this association in patients with a specific type of cancer. This study seeks to assess potential relationships between SDB severity and aggressiveness markers of cutaneous melanoma. METHODS: Four hundred and forty-three patients with a diagnosis of melanoma underwent a sleep study within 6 months of diagnosis. General demographics were collected, along with melanoma characteristics and polygraphic parameters consisting of the apnea-hypopnea index (AHI) and indices of both continuous and intermittent night-time oxyhemoglobin desaturation (DI4%). An exploration of independent relationships between SDB and various objective melanoma aggressiveness markers (Breslow index, presence of ulceration, presence of regression, mitotic index, stage of severity, damage to the sentinel lymph, and spreading of the melanoma) was performed. RESULTS: Patients in the upper tertiles of AHI or DI4% were 1.94 (95% CI, 1.14-3.32; P = .022) and 1.93 (95% CI, 1.14-3.26; P = .013) times more likely, respectively, to present with aggressive melanoma (Breslow index > 1 mm) than those in the lowest tertiles of these sleep attributes after adjustment for age, sex, tumor location, and BMI. This association was particularly prominent among patients < 56 years of age with Breslow index > 2 mm. The presence of the additional markers of aggressiveness was also associated with higher AHI and DI4% values. CONCLUSIONS: The severity of SDB was independently associated with greater aggressiveness of cutaneous melanoma, particularly among younger patients.

The inhibitory effect of leptin on angiotensin II-induced vasoconstriction in vascular smooth muscle cells is mediated via a nitric oxide-dependent mechanism.

Leptin inhibits the contractile response induced by angiotensin (Ang) II in vascular smooth muscle cells (VSMCs) of the aorta. We studied in vitro and ex vivo the role of nitric oxide (NO) in the effect of leptin on the Ang II-induced vasoconstriction of the aorta of 10-wk-old Wistar rats. NO and nitric oxide synthase (NOS) activity were assessed by the Griess and (3)H-arginine/citrulline conversion assays, respectively. Stimulation of inducible NOS (iNOS) as well as Janus kinases/signal transducers and activators of transcription (JAK/STAT) and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways were determined by Western blot. The contractile responses to Ang II were evaluated in endothelium-denuded aortic rings using the organ bath system. Changes in intracellular Ca(2+) were measured in VSMCs using fura-2 fluorescence. Leptin significantly (P < or = 0.01) stimulated NO release and NOS activity in VSMCs. Leptin's effect on NO was abolished by the NOS inhibitor, N(G)-monomethyl l-arginine, or the iNOS selective inhibitor L-N(6)-(1-iminoethyl)-lysine. Accordingly, leptin increased iNOS protein expression, with a comparable time course with that of NO production and NOS activity. Leptin also significantly increased STAT3 (P < or = 0.01) and Akt (P < or = 0.001) phosphorylation. Moreover, either the JAK2 inhibitor, AG490, or the PI3K inhibitor, wortmannin, significantly (P < or = 0.05) abrogated the leptin-induced increase in iNOS protein. Finally, both N(G)-monomethyl L-arginine and L-N(6)-(1-iminoethyl)-lysine inhibitors completely blunted (P < or = 0.001) the leptin-mediated inhibition of the Ang II-induced VSMC activation and vasoconstriction. These findings suggest that the endothelium-independent depressor action of leptin is mediated by an increase of NO bioavailability in VSMCs. This process requires the up-regulation of iNOS through mechanisms involving JAK2/STAT3 and PI3K/Akt pathways.

Phagocytic NADPH oxidase overactivity underlies oxidative stress in metabolic syndrome.

Oxidative stress plays a critical role in the pathogenesis of atherosclerosis in patients with metabolic syndrome. This study aimed to investigate whether a relationship exists between phagocytic NADPH oxidase activity and oxidative stress and atherosclerosis in metabolic syndrome patients. The study was performed in 56 metabolic syndrome patients (metabolic syndrome group), 99 patients with one or two cardiovascular risk factors (cardiovascular risk factor group), and 28 healthy subjects (control group). NADPH oxidase expression and activity was augmented (P < 0.05) in metabolic syndrome compared with cardiovascular risk factor and control groups. Insulin was enhanced (P < 0.05) in metabolic syndrome patients compared with cardiovascular risk factor and control groups and correlated with NADPH oxidase activity in the overall population. Insulin stimulated NADPH oxidase activity; this effect was abolished by a specific protein kinase C inhibitor. Oxidized LDL and nitrotyrosine levels and carotid intima-media thickness were increased (P < 0.05) in the metabolic syndrome group compared with cardiovascular risk factor and control groups and correlated with NADPH oxidase activity in the overall population. These findings suggest that phagocytic NADPH oxidase overactivity is involved in oxidative stress and atherosclerosis in metabolic syndrome patients. Our findings also suggest that hyperinsulinemia may contribute to oxidative stress in metabolic syndrome patients through activation of NADPH oxidase.

A novel CYBA variant, the -675A/T polymorphism, is associated with essential hypertension.

OBJECTIVE: Oxidative stress is implicated in hypertension and the NADPH oxidase systems constitute the main source of superoxide in vascular wall. We searched for new polymorphisms within the CYBA promoter, the human gene that encodes the p22phox protein, and studied their potential association with essential hypertension. DESIGN: A case-control study in a random sample of the general population. METHODS: CYBA polymorphisms were determined by restriction fragment length polymorphism and allelic discrimination. NADPH oxidase activity was quantified in phagocytic cells by chemiluminescence. RESULTS: We identified three novel polymorphisms, at positions -852, -675 and -536 from the ATG codon. Only the -675(A/T) polymorphism associated with essential hypertension. The prevalence of the TT genotype and the T allele frequency were significantly higher (P < 0.05) in hypertensives than in normotensives. Furthermore, TT hypertensives exhibited higher (P < 0.05) systolic blood pressure values than TA/AA hypertensives. Increased phagocytic NADPH oxidase activity was observed in TT subjects compared to TA and AA individuals (P < 0.05). Enhanced carotid intima-media thickness, a surrogate marker of atherosclerosis, was found in TT subjects compared to TA and AA individuals (P < 0.05). Finally, mutagenesis experiments demonstrated a functional role of this polymorphism on the CYBA promoter activity. CONCLUSION: The -675 (A/T) CYBA polymorphism may be a novel genetic marker associated with essential hypertension. Furthermore, TT subjects exhibit features of NADPH oxidase-mediated oxidative stress and asymptomatic atherosclerosis.

Mechanisms underlying the cardiac antifibrotic effects of losartan metabolites.

Excessive myocardial collagen deposition and cross-linking (CCL), a process regulated by lysyl oxidase (LOX), determines left ventricular (LV) stiffness and dysfunction. The angiotensin II antagonist losartan, metabolized to the EXP3179 and EXP3174 metabolites, reduces myocardial fibrosis and LV stiffness in hypertensive patients. Our aim was to investigate the differential influence of losartan metabolites on myocardial LOX and CCL in an experimental model of hypertension with myocardial fibrosis, and whether EXP3179 and EXP3174 modify LOX expression and activity in fibroblasts. In rats treated with NG-nitro-L-arginine methyl ester (L-NAME), administration of EXP3179 fully prevented LOX, CCL and connective tissue growth factor (CTGF) increase, as well as fibrosis, without normalization of blood pressure (BP). In contrast, administration of EXP3174 normalized BP and attenuated fibrosis but did not modify LOX, CCL and CTGF. In TGF-ß1-stimulated fibroblasts, EXP3179 inhibited CTGF and LOX expression and activity with lower IC50 values than EXP3174. Our results indicate that, despite a lower antihypertensive effect, EXP3179 shows higher anti-fibrotic efficacy than EXP3174, likely through its ability to prevent the excess of LOX and CCL. It is suggested that the anti-fibrotic effect of EXP3179 may be partially mediated by the blockade of CTGF-induced LOX in fibroblasts.

l-Carnitine ameliorates the oxidative stress response to angiotensin II by modulating NADPH oxidase through a reduction in protein kinase c activity and NF-κB translocation to the nucleus.

l-Carnitine (LC) exerts beneficial effects in arterial hypertension due, in part, to its antioxidant capacity. We investigated the signalling pathways involved in the effect of LC on angiotensin II (Ang II)-induced NADPH oxidase activation in NRK-52E cells. Ang II increased the generation of superoxide anion from NADPH oxidase, as well as the amount of hydrogen peroxide and nitrotyrosine. Co-incubation with LC managed to prevent these alterations and also reverted the changes in NADPH oxidase expression triggered by Ang II. Cell signalling studies evidenced that LC did not modify Ang II-induced phosphorylation of Akt, p38 MAPK or ERK1/2. On the other hand, a significant decrease in PKC activity, and inhibition of nuclear factor kappa B (NF-kB) translocation, were attributable to LC incubation. In conclusion, LC counteracts the pro-oxidative response to Ang II by modulating NADPH oxidase enzyme via reducing the activity of PKC and the translocation of NF-kB to the nucleus.

Increased phagocytic NADPH oxidase activity associates with coronary artery calcification in asymptomatic men.

Vascular calcification is a common feature in atherosclerosis and associates with cardiovascular events. Oxidative stress may be involved in the pathogenesis of vascular calcification. Previous studies have shown that the phagocytic NADPH oxidase is associated with atherosclerosis. The objective of the present study was to investigate the association between phagocytic NADPH oxidase-mediated superoxide production and coronary artery calcium (CAC). NADPH oxidase-mediated superoxide production was determined by chemiluminescence and CAC by computed tomography in 159 asymptomatic men free of overt clinical atherosclerosis. Multivariate linear regression analyses were used to assess the relationship between CAC and NADPH oxidase-mediated superoxide production. Compared with individuals in the lowest score of CAC (= 0 Agatston units), those in the upper score (>400 Agatston units) showed higher superoxide production (p < 0.05). In correlation analysis, superoxide production positively (p < 0.01) correlated with CAC, which in multivariate analysis remained significant after adjusting for age, HDL-cholesterol, triglycerides, body mass index, smoking, arterial hypertension and diabetes mellitus. In conclusion, in a population of men without clinically overt atherosclerotic disease, increased NADPH oxidase-mediated superoxide production associated with enhanced CAC. Albeit descriptive, these findings suggest a potential involvement of phagocytic NADPH oxidase-mediated oxidative stress in CAC.

Lysyl Oxidase Induces Vascular Oxidative Stress and Contributes to Arterial Stiffness and Abnormal Elastin Structure in Hypertension: Role of p38MAPK.

AIMS: Vascular stiffness, structural elastin abnormalities, and increased oxidative stress are hallmarks of hypertension. Lysyl oxidase (LOX) is an elastin crosslinking enzyme that produces H2O2 as a by-product. We addressed the interplay between LOX, oxidative stress, vessel stiffness, and elastin. RESULTS: Angiotensin II (Ang II)-infused hypertensive mice and spontaneously hypertensive rats (SHR) showed increased vascular LOX expression and stiffness and an abnormal elastin structure. Mice over-expressing LOX in vascular smooth muscle cells (TgLOX) exhibited similar mechanical and elastin alterations to those of hypertensive models. LOX inhibition with ß-aminopropionitrile (BAPN) attenuated mechanical and elastin alterations in TgLOX mice, Ang II-infused mice, and SHR. Arteries from TgLOX mice, Ang II-infused mice, and/or SHR exhibited increased vascular H2O2 and O2.- levels, NADPH oxidase activity, and/or mitochondrial dysfunction. BAPN prevented the higher oxidative stress in hypertensive models. Treatment of TgLOX and Ang II-infused mice and SHR with the mitochondrial-targeted superoxide dismutase mimetic mito-TEMPO, the antioxidant apocynin, or the H2O2 scavenger polyethylene glycol-conjugated catalase (PEG-catalase) reduced oxidative stress, vascular stiffness, and elastin alterations. Vascular p38 mitogen-activated protein kinase (p38MAPK) activation was increased in Ang II-infused and TgLOX mice and this effect was prevented by BAPN, mito-TEMPO, or PEG-catalase. SB203580, the p38MAPK inhibitor, normalized vessel stiffness and elastin structure in TgLOX mice. INNOVATION: We identify LOX as a novel source of vascular reactive oxygen species and a new pathway involved in vascular stiffness and elastin remodeling in hypertension. CONCLUSION: LOX up-regulation is associated with enhanced oxidative stress that promotes p38MAPK activation, elastin structural alterations, and vascular stiffness. This pathway contributes to vascular abnormalities in hypertension. Antioxid. Redox Signal. 27, 379-397.

The inhibitory effect of leptin on angiotensin II-induced vasoconstriction is blunted in spontaneously hypertensive rats.

OBJECTIVE: Leptin attenuates the angiotensin II-induced increase of cytosolic calcium ([Ca2+]i) and vasoconstriction in the aorta of normotensive Wistar rats. To determine whether these effects may be altered in hypertension, we assessed the effect of leptin on angiotensin II-induced vascular response in the aorta of 10-week-old spontaneously hypertensive rats (SHR). METHODS: Contractile responses to angiotensin II (100 nmol/l) in the presence of different concentrations of leptin (0.1, 1, 10, 100 nmol/l) were evaluated in isolated aortic rings by the organ bath system. [Ca2+]i was measured in vascular smooth muscle cells (VSMCs) using Fura-2 fluorescence. The expression of the short (OB-Ra) and long (OB-Rb) isoforms of the leptin receptor in VSMCs was evaluated by real-time reverse transcriptase-polymerase chain reaction and western-blot analysis. RESULTS: Circulating leptin concentrations were increased in SHR. Serum metabolic parameters, including glucose, insulin, total cholesterol and triglyceride levels, were also elevated in SHR. Leptin did not modify the angiotensin II-induced vasoconstriction in SHR either in intact or endothelium-denuded aortic rings. In addition, leptin was not able either to diminish the angiotensin II-induced the peak rise of [Ca2+]i or to accelerate the recovery rate to basal calcium levels in VSMCs from SHR. However, OB-Ra and OB-Rb mRNA and protein expression were increased in SHR VSMCs. CONCLUSIONS: The lack of effect of leptin on angiotensin II-induced contraction in the aorta of SHR is due to an impaired handling of [Ca2+]i in VSMCs. Hyperleptinemia and overexpression of OB-R in VSMCs could be compensatory mechanisms against VSMC leptin resistance in genetically hypertensive rats.

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.